Sam68, a nuclear RNA-binding protein, is a major substrate of the Src tyrosine kinase in mitotic cells. In addition to a tyrosine-rich C-terminal region, Sam68 also has six poly-proline (SH3-binding) sites, many of which are located in an amino-terminal region. Sam68 appears to act as an adaptor protein, associating with many SH2- and SH3-containing signal-transducing proteins (Richard et al., Mol. Cell. Biol. 15:186-197, 1995). Here we describe a novel 55kDa protein, Salpalpha, which has sequence similarity to Sam68 throughout its length. Salpalpha lacks the amino-terminal region found in Sam68, and has only a single poly-proline site, which binds the SH3 domain of the p85 subunit of PI 3-kinase. Salpalpha is tyrosine-phosphorylated when expressed in Rous sarcoma virus-infected chicken embryo fibroblasts (RSV-CEF); unlike Sam68, however, Salpalpha does not co-precipitate with v-Src. Salpbeta, an alternatively spliced isoform lacking the C-terminal tyrosine-rich region, is also tyrosine-phosphorylated in RSV-CEF, and also binds the SH3 domain of p85. We further show that expression of either Salpalpha or Salpbeta down-regulates the expression of Sam68 in CEF, and arrests the growth of these cells. Our results suggest that Salp may function as a negative regulator of cell growth.