The role and expression of ROCKI and ROCKII in human breast cancer was investigated and the effect on clinical outcome assessed. ROCK knockdown cells (MDA-MB-231DeltaROCKI and MDA-MB-231DeltaROCKII were tested for their in vitro invasiveness, motility and in vivo tumour growth. Samples of fresh frozen breast tumour tissue (n=113) and normal background tissue (n=30) were processed for immunohistochemical and quantitative RT-PCR analyses. MDA-MB-231DeltaROCKI and MDA-MB-231DeltaROCKII cells showed significantly decreased invasiveness compared with control cells (mean +/- SEM 4.33+/-0.84 MDA-MB-231DeltaROCKI vs. 18.4+/-1.4 control, p<0.001; 6.8+/-1.2 MDA-MB-231DeltaROCKII vs. 18.4+/-1.4 control, p<0.001). Similarly, both exhibited reduced motility compared with control cells (p<0.001) and lost their response to HGF. Importantly, no significant difference existed between knockdown and control cells in in vivo tumour growth. ROCKI was significantly higher in human mammary tumours than normal background tissue (2.9+/-1.1 vs. 0.29+/-0.13, p=0.023), although expression of ROCKII was fairly consistent in both (2050+/-646 vs. 2303+/-2079). ROCKI expression was greater in patients who died from breast cancer than in patients who remained disease free (11.6+/-7.1 vs. 1.95+/-0.95). Higher levels of ROCKI were associated with increased grade (0.95+/-0.73 grade-1; 2.11+/-1.72 grade-2; and 4.06+/-1.99 grade-3). Levels of ROCKI, but not ROCKII, were significantly correlated with overall survival of patients (p=0.004, Univariate analysis, median follow-up 120 month). These results show that ROCKI and possibly ROCKII are key factors in regulation of motility/invasion of breast cancer cells. This, together with significant correlation between ROCKI and poor outcome in clinical breast cancer, indicates that it is a potential target in human breast cancer.