Erythropoietin (EPO) is the primary regulator of mammalian erythropoiesis, providing a proliferative and differentiative signal to the early EPO-responsive erythroid progenitors, burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid, as well as to later EPO-responsive erythroid progenitors. EPO is secreted by the kidney in response to hypoxia and anemia. There is an extensive biological crossreactivity between human EPO and the EPOs of other mammals. Necas et al. have reported that this crossreactivity may not include the guinea pig (Cavia porcelllus). Because the specificity of the guinea pig's erythropoietic responses may be of biological significance, we compared guinea pig hypoxic serum with mouse (m) and human (h) recombinant (r) EPOs for their ability to induce erythroid progenitor proliferation and differentiation in semisolid cultures. Guinea pig bone marrow mononuclear cells (BMMCs) formed BFU-E colonies in response to guinea pig hypoxic serum, rhEPO, or rmEPO in a dose-dependent fashion. Neither human nor mouse BMMCs responded to guinea pig hypoxic serum; however, guinea pig hypoxic serum exerted no inhibitory effect on human or mouse in vitro erythroid differentiation in the presence of rhEPO or rmEPO. The intensity of the EPO band on Western blotting analysis of guinea pig hypoxic serum was significantly greater than in nonhypoxic serum. This suggests that guinea pig erythropoiesis is mediated by EPO and stimulated by hypoxia in a fashion similar to that observed in human and mouse erythropoiesis. Furthermore, guinea pig EPO did not stimulate human or mouse erythroid differentiation in vitro, whereas guinea pig erythroid progenitors could be stimulated by human or mouse EPO, suggesting structural differences in guinea pig EPO and EPO receptor (EPOR) compared with human or mouse EPO and EPOR. These differences probably evolved after the guinea pig's ancestors diverged from myomorph rodents. Further characterization of the guinea pig EPO and EPOR should facilitate our understanding of the interaction between EPO and EPOR.