In the biosynthesis of the tripyrrolic pigment prodigiosin, PigB is a predicted flavin-dependent oxidase responsible for formation of 2-methyl-3-amylpyrrole (MAP) from a dihydropyrrole. To prove which dihydropyrrole is the true intermediate, both possibilities, 5a (resulting from transamination of the aldehyde of 3-acetyloctanal) and 6 (resulting from transamination of the ketone), were synthesised. Only 5a restored pigment production in a strain of Serratia sp. ATCC 39006 blocked earlier in MAP biosynthesis. PigB is membrane-associated and inactive when its transmembrane domain was deleted, but HapB, its homologue in Hahella chejuensis, lacks the transmembrane domain and is active in solution. Two colorimetric assays for PigB and HapB were developed, and the HapB-catalysed reaction was kinetically characterised. Ten analogues of 5a were synthesised, varying in the C2 and C3 side-chains, and tested as substrates of HapB in vitro and for restoration of pigment production in Serratia ΔpigD in vivo. All lengths of side-chain tested at C3 were accepted but only short side-chains at C2 were accepted. The knowledge that 5a is an intermediate in prodigiosin biosynthesis and the ease of synthesis of analogues of 5a makes a range of prodigiosin analogues readily available by mutasynthesis.