Argimycins P are a recently identified family of polyketide alkaloids encoded by the cryptic gene clusterarpofStreptomyces argillaceus. These compounds contain either a piperideine ring, or a piperidine ring which may be fused to a five membered ring, and a polyene side chain, which is bound in some cases to anN-acetylcysteine moiety. Thearpcluster consists of 11 genes coding for structural proteins, two for regulatory proteins and one for a hypothetical protein. Herein, we have characterized the post-piperideine ring biosynthesis steps of argimycins P through the generation of mutants inarpgenes, the identification and characterization of compounds accumulated by those mutants, and cross-feeding experiments between mutants. Based in these results, a biosynthesis pathway is proposed assigning roles to everyarpgene product. The regulation of thearpcluster is also addressed by inactivating/overexpressing the positive SARP-likearpRIand the negative TetR-likearpRIItranscriptional regulators and determining the effect on argimycins P production, and through gene expression analyses (reverse transcription PCR and quantitative real-time PCR) ofarpgenes in regulatory mutants in comparison to the wild type strain. These findings will contribute to deepen the knowledge on the biosynthesis of piperidine-containing polyketides and provide tools that can be used to generate new analogs by genetic engineering and/or biocatalysis.