In several plant species, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase consists of two isoforms that are produced by alternative splicing of a pre-mRNA. Two forms of activase corresponding to the longer, redox-regulated alpha and the shorter, beta forms were detected immunologically in cotton (Gossypium hirsutum L.) leaves, but their N-termini differed in 4 of 14 residues. The cDNAs for the alpha and beta forms of cotton activase diverged throughout the translated and 3'-untranslated regions, including variations that accounted for the differences in N-terminal amino acid sequence. Analysis of genomic DNA confirmed that separate genes encoded the alpha and beta forms of cotton activase. Separate activase genes were also detected in diploid species of cotton containing the different progenitor genomes of the cultivated allotetraploid, indicating that the occurrence of separate alpha- and beta-form genes in cotton predates the merger of the diploid genomes. The deduced amino acid sequences of the two forms of cotton activase exhibited 84% identity and both forms were active after expression in Escherichia coli. The recombinant alpha and beta forms exhibited similar affinities for ATP and only minor differences in thermotolerance, but their ATPase specific activities differed. The results show for the first time a plant species with two forms of activase that are structurally and functionally equivalent to the alternatively spliced alpha and beta forms in other plants, but that are encoded by separate genes. That cotton still expresses both forms of activase, even without alternative splicing, suggests that each form has a required function in photosynthesis.