Our laboratory previously reported that bovine thymocytes and fibroblasts proliferated only in response to bovine IL-1, whereas proliferation of murine thymocytes was augmented equally well by murine, human, and bovine IL-1. In this study, we used direct and competitive receptor binding assays to determine whether differential binding of homologous versus heterologous IL-1 accounts for the species-specific response of bovine fibroblasts. Our results demonstrated that bovine and human fibroblasts bound homologous IL-1 with high affinity and heterologous IL-1 with lower affinity. In contrast, murine fibroblasts bound both homologous and heterologous IL-1 with high affinity. Because IL-1 and the human IL-1 receptor antagonist (IL-1ra) both bind to type 1 IL-1 receptors, we also determined whether the IL-1ra demonstrated receptor binding properties similar to human IL-1 for bovine, human, and murine fibroblasts. To our surprise, the human IL-1ra bound equally well to bovine, human and murine fibroblasts. We used this characteristic of the IL-ra to perform affinity cross-linking analysis of the IL-1 receptors on bovine, human and murine fibroblasts. These comparisons demonstrated the IL-1 receptors on bovine and human fibroblasts have similar molecular sizes (M(r) 73 kDa and M(r) kDa, respectively), whereas, IL-1 receptors on murine fibroblasts have an estimated molecular size of M(r) 88 kDa. These data demonstrate that IL-1 receptors on bovine fibroblasts preferentially bind homologous IL-1, but bovine fibroblasts do not discriminate binding by the human IL-1ra. In contrast, murine fibroblast IL-1 receptors bind heterologous IL-1 with high affinity.