Activation of Rubisco, the primary carboxylating enzyme in photosynthesis, is promoted in vivo by the enzyme Rubisco activase. As a first step in investigating Rubisco activation in cotton, we screened a cotton (Gossypium hirsutum L.) cDNA library to isolate the cotton activase (rca) gene. Restriction and sequence analysis of positive clones indicated that there were three classes of cotton rca cDNAs. One of these classes encodes for a mature protein of 380 amino acids (42357 Da), while the other two encode for mature proteins of 419 amino acids (46590 Da). These results were consistent with Southern analysis which indicated that there are multiple cotton activase genes and with Western blot analysis which showed the presence of two activase polypeptides in cotton leaf tissue. The deduced amino acid sequences of the cotton rca clones shared 80 to 89% identity with activases from other higher plants. One of the cotton activase clones was overexpressed in E. coli, enabling the production of large amounts of soluble cotton activase protein for functional analysis.