The pharmacokinetics of several proteins were investigated using two different assays. A 23 kDa recombinant protein fragment of bactericidal/permeabilityincreasing protein (rBPI,,) was radiolabeled with 1251 using Iodo-beads@ and administered to rats. Plasma samples were collected and assayed for 'Z51-rBPI,3 by radioactivity. In a separate experiment, rBPI,, was administered to rats and plasma samples were assayed for rBPI,, by ELISA. The clearance determined from plasma concentrations of 1251-rBP123 measured by radioactivity was about 2.5-fold lower than that of rBPI,, determined by ELISA. In addition, the steady state volumes of distribution and mean residence times of 1251-rBPIz3 measured by radioactivity were four-fold and 10-fold greater, respectively, compared to those measured by the ELISA method. By studying several proteins with a range of molecular weights, we found that the pharmacokinetics of proteins below about 60 kDa were different when assayed by radioactivity or ELISA, but those of proteins with molecular weights of at least 80 kDA revealed only minor differences. To determine which assay method yielded the correct plasma pharmacokinetic profile, rBPIz3 was metabolically labeled with 35S-methionine and administered to rats, and plasma samples were assayed by radioactivity. The concentration-time profile assessed by this method was very close to that determined by ELISA. Exposing rBPIZ3 to chloramine-T (the oxidant used in the iodination process) and measuring its plasma concentration by ELISA revealed pharmacokinetics similar to those of the iodinated protein measured by radioactivity. In contrast, radiolabeling rBP12, using iodinated Bolton-Hunter reagent (which avoids exposing the protein to oxidant), and measuring '2SI-rBP12! by radioactivity, yielded pharmacokinetics that were similar, although not identical, to the pharmacokinetics of rBPI,, measured by ELISA. Thus, our data suggest that directly iodinating low-molecular-weight proteins by oxidation procedures alters their clearance from the blood, preventing reliable determination of pharmacokinetic parameters.