src kinase activity is elevated in some human tumors, including breast and colon cancers. The precise cellular function of the src family kinases is not clearly understood, but they appear to be involved in numerous signaling pathways. We studied the effects of PD173955, a novel src family-selective tyrosine kinase inhibitor, on cancer cell lines and found that it has significant antiproliferative activity due to a potent arrest of mitotic progression. The mitotic block occurs after chromosome condensation in prophase, before spindle assembly and without loss of cyclin A and B kinase activities. This effect is seen in cancer cell lines of all types with low or high activities of src kinases as well as in untransformed cell lines. In MDA-MB-468 breast cancer cells, this drug produces a rapid inhibition of cellular src and yes kinase activities as well as suppression of the mitotic hyperactivity of these kinases. This compound defines a novel class of antimitotic drugs that work through inhibition of src kinases and possibly other protein kinases that are required for progression through the initial phases of mitosis.