To elucidate the properties of the Src-family member Bsk/Iyk, NIH3T3 cells were transfected with wild-type Bsk/Iyk or Bsk/Iyk carrying Y497F, Y504F or Y497/504F mutations. These positions are putatively homologous to tyr-527 in Src. The Bsk/IykY497/504F cells displayed a decreased cell growth rate, parallelled by an augmentation of the fraction of cells in G1-phase. The Bsk/IykY497/504F double-mutation decreased the [3H]thymidine incorporation. No effects on NIH3T3 cell growth could be seen in cells expressing wild-type Bsk/Iyk or the other Bsk/Iyk mutants. In vitro kinase reactions performed on immunoprecipitates from NIH3T3 cells expressing wild-type or mutated Bsk/Iyk revealed increased relative [32P]-incorporation into Bsk/Iyk isoforms containing the Y504F and Y497/504F mutations compared with wild-type Bsk/Iyk. The Y497F and Y497/504F mutations elevated the proportion of [32P]-incorporation into a 57 kDa Bsk/Iyk product relative to that into the 60 kDa isoform. The Y497F Bsk/Iyk mutant not only increased the relative amount of p57 Bsk/Iyk but also transferred this isoform to the nuclear subcellular fraction. The results suggest that Bsk/Iyk has unique regulatory properties, and that this kinase might serve a role in inhibiting cell replication.