We have measured endogenous and induced rates of 70-kD, 89-kD, and 110-kD heat shock proteins in highly pure G1-, S-, or G2-M phase fractions of Chinese hamster fibroblasts (CHO) separated by fluorescence-activated cell sorting (FACS). Relative rates of synthesis of all three polypeptides as measured by two-dimensional gel electrophoresis were similar throughout the cell cycle, and therefore, endogenous levels were unlikely to explain the thermal sensitivity of S-phase cells. Distinct heterogeneity in induced rates of these polypeptides was noted in all phase fractions. Enhanced rates of 70-kD polypeptide were measured in S and G2-M as compared to G1 following heat shock. Little increase in either the 89-kD or 110k-kD heat shock proteins was observed in heated G1 cells. This heterogeneity in induced rates of synthesis was in contrast to the similarity in thermal tolerance expression kinetics between each phase. Finally, enhanced synthesis of these polypeptides appeared unrelated to regulation of either heat-induced cell cycle delay or to the resumption of phase-specific progression after heat shock as measured by simultaneous flow cytometric measurement of incorporated BrdUrd and DNA content.