Identification of tumor suppressor gene loci in rodent cell culture systems has relied upon the use of somatic cell hybridization studies. Although normal rodent fibroblasts are capable of suppressing the tumorigenicity of a variety of tumor cells, the lack of complementation in tumor cell x tumor cell hybrids has left the possibility that a single tumor suppressor gene may be responsible for tumor suppression in a particular rodent cell culture system. Using this same approach, we found no evidence for complementation resulting in suppression of the transformed phenotype when three viral oncogene-transformed Syrian hamster embryo (SHE) cell lines and one spontaneously transformed baby hamster kidney (BHK) cell line were fused to benzo[a]pyrene-transformed SHE cells (BP6T-M3). However, v-src oncogene-transformed cell line (srcT) x BP6T-M3 hybrids did demonstrate limited suppression of the transformed phenotype, suggesting at least two complementing tumor suppressor genes in this system. We were able to confirm and extend this finding using another experimental approach with preneoplastic hamster cell lines that are immortal in culture but nontumorigenic in nude mice. We propose that fusion of these preneoplastic cells to various tumor cells may reveal tumor suppressor genes not evident in the tumor cell x tumor cell complementation studies. Subclones of two nontumorigenic, immortal hamster cell lines, 10W and DES4, displayed differing abilities to suppress BP6T-M3 cells in somatic cell hybrids, as quantitated by the ability of the hybrid cells to form colonies in soft agar. With a panel of preneoplastic hamster cell x BP6T-M3 hybrids, a distinct pattern of suppression or expression of the transformed phenotype was observed. Marked differences in this pattern were seen when the same 10W and DES4 subclones were fused to other hamster fibrosarcoma cell lines, indicating different tumor suppressing activities of multiple tumor suppressor genes. Analysis of this data suggests that as few as three or as many as six different tumor suppressor genes may be active in the Syrian hamster embryo cell culture system. Thus, this system may provide a useful model for identifying and studying the effects and regulation of a number of different tumor suppressor genes for fibrosarcomas.