The fine structure of high and low-yield mutants of Penicillium chrysogenum producing 10,000 and 100 units of benzylpenicillin was compared. The cells of both mutants showed typical eukaryotic ultrastructure. The Golgi vesicles, present in largest number in cells of high-yield mutant, fuse with the cell membrane and play an important role in the transport of benzylpenicillin from the cytoplasm to the cell environment. Benzylpenicillin was localized in cells of the high-yield mutant by means of enzymatical and immunological methods. The results indicate that benzylpenicillin is stored in the vesicles of the Golgi apparatus. The Golgi vesicles isolated from the protoplasts of high-yield mutant showed activities of enzymes of the pathway of benzylpenicillin biosynthesis i.e., delta-/L-alpha-aminoadipyl/-L-cysteinyl-D-valine synthetase, isopenicillin N synthetase, phenylacetyl: coenzyme A ligase, and acyl-exchange activity. Cell-free biosynthesis of antibiotic by the native Golgi vesicles was investigated in a well-defined reaction mixture. The native Golgi vesicles produced antibiotic in amount corresponding to 320 nmol.mg protein-1.h-1. The activity yield of the calcium alginate immobilized Golgi vesicles was 44%. Moreover, a hypothetical scheme for localization of the enzymes of pathway of benzylpenicillin biosynthesis in the cells of high-yield mutant is presented.