The present study characterizes constitutively expressed rat testicular interleukin-1alpha (IL-1alpha) proteins. IL-1 bioactivity of crude testis protein was completely neutralized by IL-1alpha antiserum, IL-1 receptor antagonist, and soluble type I IL-1 receptor. Upon non-denaturating gel permeation chromatography, bioactive IL-1 eluted at molecular sizes of 45, 31, and 17kDa and at charges of pH 5.7 and 6.0 after chromatofocusing. SDS-PAGE/Western blot analysis of proteins extracted from whole testis, seminiferous tubules, interstitial, and seminiferous tubule fluids all demonstrated IL-1alpha immunoreactivity at 45, 24, and 19kDa. Activated macrophages and tissue proteins from endotoxin treated rats showed immunoreactive 31 and 19kDa IL-1alpha. The results indicate that the testis produces three isoforms of IL-1alpha proteins that are secreted into the interstitial compartment and tubular lumen where they may exert paracrine functions. The testicular IL-1alpha isoforms may represent posttranslationally modified precursor, mature IL-1alpha, and a 24-kDa alternate splice form.