To investigate the deuterium isotope effect on the biological activity of penicillin G, this compound was chemically synthesized by first obtaining phenylacetyl-&chloride from phenylacetic-d,-acid and thionyl chloride, followed by condensation of the acid chloride with 6-aminopenicillanic acid. The penicillin was isolated as the 1-ethylpiperidine salt. Yield of the deuteriopenicillin was approximately 60 when a 2 : 1 (6-aminopenicillanic acid-acid chloride) molar ratio was used. The identity of the deuteriated penicillin was confirmed using several physical constants. From NMR data, the presence of deuterium in the benzyl moiety was found to be greater than 95 atom %. The biological activity of the deuteriopenicillin was compared to that of the protioanalog by a turbidimetric assay procedure using Staphylococcus aureus. The results of the biological assay indicate that a significant deuterium isotope effect operates in the antistaphylococcal action of benzylpenicillin. With the test organism chosen, the ratio of the antibiotic potencies was 125 % H/D. Keyphrases 0 Deuteriopenicillin-synthesis 0 Biological activitydeuteriopenicillin 0 Turbidimetric assay-deuteriopenicillin antimicrobial activity NMR spectroscopy-identification 0 IR spectrophotometry-identification