We have previously shown that functional ribulose bisphosphate carboxylase (RuBPCase, rbc) genes in Alcaligenes eutrophus ATCC 17707 are present both on the chromosome and on the indigenous plasmid pAE7. Here we demonstrate that the chromosomal rbc locus encodes both a large (rbcL)- and a small (rbcS)-subunit gene. A 2.3-kilobase DNA fragment containing both subunit genes was subcloned into the broad-host-range vector pRK310 to yield plasmid pAE312. This plasmid was transferred into Pseudomonas aeruginosa in which expression of both the rbcL and rbcS genes took place, as demonstrated by Western blot analysis. A high level of RuBPCase activity was observed for P. aeruginosa(pAE312), suggesting that assembly of the subunits took place. Plasmid pAE312 was mutagenized with Tn5 in Escherichia coli. Complementation of A. eutrophus RuBPCase structural gene mutants with pAE312 containing mapped Tn5 insertions allowed functional analysis of the rbc gene region. The polar effect of the Tn5 insertions suggested that the two subunit genes were cotranscribed in A. eutrophus, with rbcL located promoter proximal. Northern blot analysis of total RNA from P. aeruginosa(pAE312) confirmed cotranscription of the two subunit genes. DNA probes containing both the rbcL and rbcS genes, or fragments of each gene, all hybridized to a predominant transcript about 2.1-kilobases long. These observations indicate that the chromosomally encoded rbcL and rbcS genes of A. eutrophus constitute an operon.