Protamines are small, highly basic proteins that replace histones and testicular basic proteins during the development of mature spermatozoa, spermatogenesis. In mammals, extensive disulfide crosslinking of protamines result in the formation of a compact chromatin structure devoid of transcriptional activity. As determined by isolation and electrophoresis of protamines, only one protamine has been detected in the mature spermatozoa of most mammalian species. However, in the spermatozoa of the mouse and human, two different protamines called P1 and P2, have been found. In this report we demonstrated by electrophoretic analysis that these two protamines are also present in the spermatozoa of Microtus arvalis, Microtus agretis, Apodemus flavicollis, Apodemus sylvaticus, Clethrionomys glareolus, the Chinese and the golden hamster. However, only one protamine is found in the spermatozoa of the guinea pig, dog, bull, black monkey, and the rhesus monkey. The mammalian protamines are highly conserved during mammalian evolution. In general, the homologies on the amino acid sequence of the various mammalian protamines range from 52% to 96%. Furthermore, in the case of mouse and human protamines, the genes of the protamines are closely linked and located on chromosome 7 and 16, respectively. Accordingly, it can be assumed that both types of protamine genes have arisen by gene duplication during mammalian evolution. According to the results of an electrophoretical analysis of the mammalian protamines, the predicted point of gene duplication during evolution is deduced carefully.