We recently identified a novel human protein kinase, p160 ROCK, as a putative downstream target of the small GTPase Rho. Using the human ROCK cDNA as a probe, we isolated cDNA of two distinct, highly related sequences from mouse libraries. One encoded a mouse counterpart of human ROCK (ROCK‐I), and the other encoded a novel ROCK‐related kinase (ROCK‐II). Like ROCK/ROCK‐I, ROCK‐II also bound to GTP‐Rho selectively. ROCK‐I mRNA was ubiquitously expressed except in the brain and muscle, whereas ROCK‐II mRNA was expressed abundantly in the brain, muscle, heart, lung and placenta. These results suggest that at least two ROCK isoforms are present in a single species and play distinct roles in Rho‐mediated signalling pathways.