THE protein-tyrosine kinase activity of the proto-oncogene product p60^c-src is negatively regulated by the phosphorylation of a tyrosine residue close to the C terminus, tyrosine 527 (refs 1–11). The phosphorylation might be catalysed by a so-far-unidentified tyrosine kinase, distinct from p60^c-src (ref. 7). Recently we purified a protein-tyrosine kinase that specifically phosphorylates tyrosine 527 of p60^c-src from neonatal rat brain^8,12,13. We have now confirmed the specificity of this enzyme by using a mutant p60^c-src that has a phenylalanine instead of tyrosine 527, and cloned a complementary DNA that encodes the enzyme. The enzyme is similar to kinases of the src family in that it has two conserved regions, Src-homology regions 2 and 3, upstream of a tyrosine kinase domain. The amino-acid identity of each region is no more than 47%, however, and the enzyme lacks phosphorylation sites corresponding to tyrosines 416 and 527 of p60^c-src and has no myristylation signal. These results suggest that this protein-tyrosine kinase, which might negatively regulate p60^c-src, represents a new type of tyrosine kinase.