Mutations in src homology regions 2 and 3 of activated chicken c-src that result in preferential transformation of mouse or chicken cells. src homology regions 2 and 3 (SH2 and SH3) of proteins encoded by src and closely related genes are conserved domains believed to modulate the protein-tyrosine kinase activity of this class of proteins, perhaps through interactions with other proteins. To explore the possibility of using src mutants as probes for such interactions, we have compared mouse NIH 3T3 cells with chicken embryo fibroblasts as host cells for 24 previously described substitution and deletion mutants with lesions in the SH2- and SH3-encoding regions of a transformation-competent allele of chicken c-src. Although several of these mutants are equally competent or equally defective for transformation of the two cell types, four mutants (three of which map within SH3) preferentially transform NIH 3T3 cells, and seven mutants (all of which map within SH2) preferentially transform chicken cells. Some of the SH2 mutants least able to transform mouse cells exhibit augmented transforming activity in chicken cells. In general, the in vitro protein-tyrosine kinase activities of the mutants correlated with transforming activities. Thus, in many cases, the catalytic activity of a mutant protein depended upon the host cell in which the protein was made. Such host-dependent mutants may be especially useful reagents for biochemical and genetic studies of the src gene family. 