In metazoans, the HR1 domain, a motif found in a number of proteins including the protein kinase C-related PRKs, is responsible for an interaction with Rho-GTPases. The structural similarity between the Schizosaccaromyces pombe Pck proteins and the mammalian Rho-dependent protein kinase C-related family, has led us to investigate the relationship between the function of Rho and that of Pck1/2. Rho1 is shown to interact with the conserved N-terminal HR1 domain of Pck1/2 in vitro and in vivo. Lethal overproduction of Rho1 is neutralized by co-expression of the Pck2 HR1 domain, which by itself compromises growth when overproduced. The Pck2-Rho1 interaction has a profound effect on the steady state expression of Pck2 and this is shown to parallel the immunoprecipitated activity and phosphorylation of Pck2 at its activation loop site. It is further shown that Pck2 becomes localized at the septum, where Rho1 is also located. The results demonstrate that the Pck proteins are Rho1 effectors in fission yeast and that the HR1 domain is a universal motif for the Rho-GTPase interaction. Furthermore, the evidence supports the contention that the yeast Pck1 and Pck2 proteins are primitive protein kinases, which in vertebrates have evolved into the two distinct PKC and PRK families.