The chaperonins GroEL and Cpn60 were isolated from the cyanobacterium Synechocystis PCC 6803 and characterized. In cells grown under optimal conditions their ratio was about one to one. However, the amount of GroEL increased considerably more than that of Cpn60 in response to heat stress. The labile chaperonin oligomer required stabilization by MgATP or glycerol during isolation. Use of the E. coli mutant strain, groEL44 revealed that the functional properties of the two cyanobacterial chaperonins are strikingly different. Overexpression of cyanobacterial GroEL in the E. coli mutant strain allowed growth at elevated temperature, the formation of mature bacteriophage T4, and active Rubisco enzyme assembly. In contrast, Cpn60 partially complemented the temperature-sensitive phenotype, the Rubisco assembly defect and did not promote the growth of the bacteriophage T4. The difference in chaperone activity of the two cyanobacterial chaperonins very probably reflects the unique chaperonin properties required during the life of Synechocystis PCC 6803.