A gene, aat, encoding acyl-CoA: 6-aminopenicillanic acid acyltransferase (AAT), the last enzyme of the penicillin (Pn) biosynthetic pathway, has been cloned from the genome of Penicillium chrysogenum AS-P-78. The gene contains three introns in the 5'-region and encodes a protein of 357 amino acids with an Mr of 39,943. It complements mutants of P. chrysogenum deficient in AAT activity. The aat gene is expressed as a 1.15-kb transcript and the encoded protein appears to be processed post-translationally into two nonidentical polypeptides of 102 and 255 aa, with Mrs of 11,498 and 28,461, respectively. Three proteins of 40, 11, and 29 kDa (the last one corresponding to the previously purified AAT), were identified in extracts of P. chrysogenum. The aa sequence of the N-terminal end of the 11-kDa polypeptide matched the nucleotide (nt) sequence of the 5'-region of aat. The N-terminal end of the 29-kDa polypeptide corresponded to the sequence beginning at nt position 916 of the sequenced DNA fragment (nt 441 of aat gene). The aat gene of P. chrysogenum resembles the genes encoding Pn acylases of Escherichia coli, Proteus rettgeri and Pseudomonas sp., all of which encode two nonidentical subunits derived from a common precursor, encoded by a single open reading frame.