Since mammalian cells vary widely in their intrinsic thermoresistance, we have investigated the genetic basis underlying this phenomenon in human and rodent cell lines. Typically, human cells are considerably more resistant to killing by heat than rodent cell lines. To determine whether the heat-resistant phenotype is dominant or recessive and to locate the chromosome(s) bearing determinants for heat resistance, we have prepared hybrids of heat-resistant human HT1080 cells and heat-sensitive Chinese hamster ovary (CHO) cells to test their response to heat. For both mass hybrid cultures and individual clones, the heat response of the hybrids was similar to that of the CHO parent. Analysis by in situ hybridization revealed the presence of five to 20 human chromosomes per cell in the mass hybrids and four to eight intact chromosomes plus some fragments in individual clones isolated from the hybrid cell population. A similar result was obtained using a different human cell line, AG1522. These data suggest that heat resistance is a recessive trait. Consistent with this conclusion are the results from a study of a fusion of HT1080 to a CHO mutant, BL-10, which was found to be hypersensitive to heat-induced killing. These hybrids had a normal CHO heat response and not the more heat-resistant phenotype of HT1080 cells. Two hybrid clones, H2 and H4, from the HT1080/BL-10 fusion were studied in more detail. Both clones possess similar amounts of Mr 70,000 heat shock protein (HSP70), despite the fact that H4 contains three human chromosomes (Nos. 6, 14, and 21) which carry HSP70 genes while H2 contains only one (chromosome 6). Both hybrid cell lines have the same response to heat. Although we found a wide range of sensitivities to heat, all cell lines contained a similar amount of constitutive HSP70, suggesting that HSP70 levels per se are not the critical determinant of intrinsic heat resistance.