The existence and function of a Hsp40-Hsp70 chaperone machinery in mammalian cells in vivo was investigated. The rate of heat inactivation of firefly luciferase transiently expressed in hamster O23 fibroblasts was analyzed in cells co-transfected with the gene encoding the human Hsp40 (Ohtsuka, K. (1993) Biochem. Biophys. Res. Commun. 197, 235-240), the human inducible Hsp70 (Hunt, C., and Morimoto, R. I. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6455-6459), or a combination of both. Whereas the expression of human Hsp70 alone in hamster cells was sufficient for the protection of firefly luciferase during heat shock, expression of the human Hsp40 alone was not. Rather, this led to a small but significant increase in the heat sensitivity of luciferase. The expression of the human Hsp40 only led to heat protection when the human Hsp70 was expressed as well. Under such conditions the rate of luciferase reactivation from the heat-inactivated state was increased, but the rate of inactivation during heat shock was not affected. Using constructs that direct firefly luciferase either to the cytoplasm or to the nucleus (Michels, A. A., Nguyen, V.-T., Konings, A. W. T., Kampinga, H. H., and Bensaude, O. (1995) Eur. J. Biochem. 234, 382-389), it was demonstrated that these chaperone functions are found in both compartments. Our data provide the first evidence on how the Hsp40/Hsp70 chaperone complex acts as heat protector in mammalian cells in vivo.