We have compared the relative proportion of protamine 1 (P1) and protamine 2 (P2) bound to DNA in the sperm of a variety of eutherian mammals to obtain insight into how these two proteins interact in sperm chromatin. Gel electrophoresis (combined with microdensitometry) and high performance liquid chromatography (HPLC) were used to determine the content of the two protamines, and the identity of each protein was confirmed by amino-terminal sequencing or amino acid analysis. The sperm of all species examined contained P1, but P2 was found to be present only in certain species. Unlike the fixed ratio of core histones that package DNA into nucleosomes in all somatic cells, the proportion of P2 present in mature sperm was found to be continuously variable from 0 to nearly 80%. These results show that P1 and P2 do not interact with each other or DNA to form a discrete complex or subunit structure that is dependent upon particular P1/P2 stoichiometries. Data obtained from a number of closely and distantly related species also indicate that while the P2 content of sperm chromatin is allowed to vary over a wide range during the course of evolution, the relative proportion of P1 and P2 are tightly regulated within a genus.