The 5-aminolevulinate synthase gene (hemA) from Rhizobium meliloti was used to probe a genomic lambda bank derived from Rhodobacter sphaeroides DNA. Two phage clones were found to bear homology to the Rhizobium probe. Southern hybridization analysis of the two lambda phage clones, which we designated lambda Hem 10 and lambda Hem 12, showed that the homology to the Rhizobium hemA gene was localized to a 3.1-kb SalI fragment derived from lambda Hem 10 and a 7.0-kb SalI fragment derived from lambda Hem 12. Each of the SalI fragments was subsequently cloned into the multiple cloning site of pUC19 in both orientations relative to the lac promoter. Restriction analysis confirmed that each SalI fragment was unique. It was also shown from Southern hybridization analysis that the regions of homology within each of the R. sphaeroides restriction fragments and the Rhizobium probe were different. Further, we have tentatively concluded that each R. sphaeroides hemA gene shows a relatively low degree of homology to the other. Data obtained from in vitro transcription-translation studies in a homologous R. sphaeroides cell-free system, and complementation of hemA mutations of both Escherichia coli and R. sphaeroides by either of the putative hemA clones suggested the presence of a gene encoding 5-aminolevulinate synthase on each DNA sequence. The fact that 5-aminolevulinate synthase activity could be demonstrated in mutant strains complemented in trans with either cloned DNA fragment further supported this conclusion.