The pur cluster which encodes the puromycin biosynthetic pathway from Streptomyces alboniger was subcloned as a 13-kilobase fragment in plasmid pIJ702 and expressed in an apparently regulated manner in the heterologous host Streptomyces lividans. The sequencing of a 9.1-kilobase DNA fragment completed the sequence of pur. This permitted identification of seven new open reading frames in the order: napH, pur7, pur10, pur6, pur4, pur5, and pur3. The latter is followed by the known pac, dmpM, and pur8 genes. Nine open reading frames are transcribed rightward as a unit in opposite direction to that of the pur8 gene which is expressed as a monocistronic transcript from the right-most end. napH encodes the known N-acetylpuromycin N-acetylhydrolase. The deduced products from other open reading frames present similarities to: NTP pyrophosphohydrolases (pur7), several oxidoreductases (pur10), the putative LmbC protein of the lincomycin biosynthetic pathway from Streptomyces lincolnensis (pur6), S-adenosylmethionine-dependent methyltransferases (pur5), a variety of presumed aminotransferases (pur4), and several monophosphatases (pur3). According to these similarities and to previous biochemical work, a puromycin biosynthetic pathway has been deduced. No cluster-associated regulatory gene was found. However, both pur10 and pur6 genes contain a TTA codon, which suggests that they are translationally controlled by the bldA gene product, a specific tRNA(Leu).