The protamine P1 genes from two monotremes, platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus) were isolated after polymerase-chain-reaction amplification then cloned and sequenced. The two protamine P1 genes are of 290 bp and 311 bp for platypus and echidna, respectively, and are clearly orthologous to the published sequences of protamine P1 genes of eutherian mammals and birds. Both genes contain an intron, like the mammals and marsupials and unlike the bird P1 genes that are intronless. The deduced protein sequences from the coding areas of the platypus and echidna protamine P1 genes do not contain any cysteine residues. This absence of cysteine residues leaves the sperm nuclei susceptible to disruption in vitro by exposure to increasing ionic strength and is a characteristic of fish, birds and marsupials. In contrast, the P1 protamines of placental mammals invariably contain 6-9 cysteine residues that, as a result of the formation of intermolecular and intramolecular disulfide bridges, significantly increase the stability of the sperm nuclei that can only be disrupted following disulfide-bond cleavage. Phylogenetic analysis of the protamine P1 gene sequences indicates that the monotremes occupy a position half-way between the eutherian mammals and birds. From the DNA sequences we estimate the time of divergence of the platypus and the echidna to be around 22 million years ago. This date agrees very well with the published estimates of divergence based on other criteria.