Staurosporine (SSP) is a microbial alkaloid isolated from cultures of Streptomyces. For its ability to specifically inhibit protein kinase C and other serine/threonine and tyrosine protein kinases, SSP has been utilized recently to synchronize cells in G1 and/or G2 phases of the cell cycle. In the present paper we focus on the synchronizing properties of SSP with respect to three human normal cell types (PHA-activated peripheral blood lymphocytes, HS-68 and WI-38) and eight human tumor cell cultures (SV-40 transformed WI-38, SW48, SW480, A253, T-24, A549, MOLT-4 and HL-60). We describe procedures for maintenance and synchronization of these cells in culture. We provide the DNA/RNA flow cytometric methodology to verify cell synchronization and to evaluate the position of cell accumulation in specific phase and the level of synchrony. Synchronization in G1 is being achieved in the three normal cell lines after 24h SSP treatment with low concentrations (5–10 ng/ml) and in G2 after 24h treatment at higher SSP concentrations (50–100 ng/ml) in four of the eight tumor cell lines (A253, SW48, SW480 and A549). The other four tumor cell cultures (SV-40, WI-38, T-24, MOLT-4 and HL-60) show, at 50–100 ng/ml SSP concentration, an apparent G2 block, which is actually due to the presence of cells entering higher DNA ploidy levels. All other cell type/SSP dose combinations fail to induce cell synchronization. We also report a summary of the literature data about cell synchronization with SSP in other cell lines. Our results, together with the results from the literature, suggest that, while SSP may be useful for synchronizing normal cells in G1, its application for synchronizing tumor and/or transformed cells is limited. Critical comments follow, as well as suggestions and words of caution addressed to the future users of SSP as a cell synchronizing agent.