In most cyanobacteria, the gene rbcX is co-transcribed with the rbcL and rbcS genes that code for the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Previous co-expression studies in Escherichia coli of cyanobacterial Rubisco and RbcX have identified a chaperonin-like function for RbcX. The organization of the rbcLXS operon has, to a certain extent, precluded definitive gene function studies of rbcX in cyanobacteria. In Synechococcus PCC7942, however, rbcX is located >100 kb away from the rbcLS operon, providing an opportunity to examine the role of RbcX by insertional inactivation without interference from the Rubisco genes. Fully segregated Synechococcus PCC7942 DeltarbcX::KmR mutants were readily obtained that showed no perturbations in growth rate or Rubisco content and activity. Low amounts of rbcX transcript were detected in Synechococcus PCC7942; however, a sensitive antibody raised against purified RbcX failed to detect RbcX expression in cells exposed to different stress treatments. In contrast, co-expression studies of Rubisco assembly in E. coli showed that RbcX from Synechococcus PCC7942 and PCC7002 are functionally interchangeable and can stimulate assembly of the PCC7942 and PCC7002 Rubisco subunits. Our results indicate that Rubisco folding and assembly in Synechococcus PCC7942 may have evolved to be independent of RbcX function, apparently in contrast to other beta-cyanobacteria. We speculate that divergent evolution of the RbcL sequence may have relaxed a requirement for RbcX function in Synechococcus PCC7942 and propose a new approach for definitively isolating RbcX function in other beta-cyanobacteria.