In developing 99m Tc-based radioligands for in vivo study of cardiac adrenergic neurons, we compared the relative uptake of the new 99m Tc-labelled compound 99m Tc-FBPBAT with those of the SPET radiotracer 123 I-MIBG in rat vascular smooth muscle cells and neonatal ventricular cardiac myocytes as established in vitro models. Specificity of cellular uptake as well as the mechanisms underlying the uptake of both radiopharmaceuticals into the cardiovascular tissues were examined by pharmacological challenge experiments. Thereafter, cardiac and extracardiac accumulation of 99m Tc-FBPBAT and 123 I-MIBG were assessed in intact rats and in rats pretreated with various aand b-adrenoceptor drugs, and adrenergic reuptake blocking agents. The cellular uptake of 99m Tc-FBPBAT and 123 I-MIBG was rapid and concentration dependent, more than 90 % of the total tissue radioactivity accumulation occuring within the first 5 min. Radioactivity concentration in cardiovascular tissues following a 60-min incubation at 37ยฐC (pH 7.4) varied from 20 to 65% of the total activity per million cells ( 123 I-MIBG < 99m Tc-FBPBAT). In comparison, tissue uptake of the cardiac perfusion radiotracers 99m Tc-MIBI and 99m Tc-tetrofosmin remained relatively low (< 3%). Uptake of 99m Tc-FBPBAT was obviously lower at 4ยฐC and 20ยฐC than at 37ยฐC, while uptake of 123 I-MIBG showed only slight temperature dependence. Competitive inhibition experiments i ndicated that the uptake of 123 I-MIBG was predominantly mediated by the adrenergic uptake-I carrier, while the a 1 -adrenoceptors and in less instance b 1 -adrenoceptors were additionally involved in the uptake of 99m Tc-FBPBAT into the cardiovascular tissues. Biodistribution in rats revealed a fast clearance of both radiopharmaceuticals from blood and a higher initial uptake of 99m Tc-FBPBAT in the lung, which decreased rapidly with time (up to 55% after 60 min). On the other hand, significant uptakes and retentions of 99m Tc-FBPBAT and 123 I-MIBG were observed in heart and spleen. Radioactivity accumulation in untreated rat myocardium 15 and 60 min following intravenous injection of 99m Tc-FBPBAT accounted for 2.32 and 1.91 % of the injected dose per gram (i.d./g), respectively, compared with 3.10 and 2.21 % i.d./g in the in vivo experiment with 123 I-MIBG. Moreover, the heart uptake of 99m Tc-FBPBAT was strongly inhibited by prazosin and metoprolol, more effectively than by desipramine and more potently than in lungs and other target organs, including kidney and spleen. In comparison, the myocardial uptake of 123 I-MIBG was only effectively lowered by pretreatment with the norepinephrine transporter inhibitor desipramine. Also, 99m Tc-FBPBAT was excreted into urine and at less intensity by faeces. Urine analysis 6h p.i. revealed that more than 40% of the total excreted radioactivity was unmetabolized product. These results suggest that heart uptake of 99m Tc-FBPBAT specifically reflects binding to cardiac adrenergic ne urons. In contrast to 123 I-MIBG, the heart uptake of 99m Tc-FBPBAT appears to be mediated predominantly via the a 1 /ร 1adrenoceptor pathways. These data indicate that 99m Tc-FBPBAT like 123 I-MIBG, exhibits interesting biological characteristics which hold promise for studies in vivo of myocardial sympathetic innervation by SPET.