The variability of sperm histones was compared in two species of sea urchin. Whole sperm specific histones (SpH), were isolated from Tetrapygus niger (Arbacoida) and Parechinus angulosus (Echinoida). Individual histones were purified by chromatography on BioGel P-60 followed by reverse high pressure liquid chromatography (HPLC). The heterogeneity of each major histone type from T. niger was established from their HPLC elution patterns and further confirmed by electrophoresis in polyacrylamide gels containing 6 mM Triton X-100 combined with a transverse urea gradient (0--8 M). In T. niger, as well as in P. angulosus, a single form of SpH1 and SpH2A were found. In contrast, SpH2B was found to be heterogeneous, but represented by one major form in both species. The relatedness between both sets of histones was determined by establishing their immunological cross-reactivity. In this context, polyclonal antibodies elicited against T. niger sperm histones were assayed against individual histones from P. angulosus. From the results obtained, it emerged that histone SpH2A was the more closely related protein between these two species, followed by histone SpH1. In contrast, histone SpH2B was found to be only moderately related. These results confirm that SpH2A did not co-evolve with SpH2B, as was predicted for most species.