We have characterized for the first time the proteins of two different species of palaeognathous birds, Struthio camelus australis (ostrich) and Nothoprocta perdicaria sanborni (Chilean tinamou). Similar to what had been previously reported in neognaths, the electrophoretic mobility, amino acid composition, and primary structure of the main protamine (P-II) component of these two species of birds are similar. However, in contrast to neognathous birds, the protamines from paleognaths display a higher electrophoretic mobility and a significantly different amino acid composition and protein sequence. The sperm and the main protamine component P-II from the ostrich reveal structural and compositional characteristics intermediate between neognathous birds and reptiles. The marked differences between the protamines and sperm structure of neognaths and paleognaths provide support to a phylogenetic relationship between neornithine birds in which these two groups represent two separate phylogenetic lines. Furthermore, these results shed some additional light on the controversial origin of birds. They provide further molecular support to the fossil record that suggests that reptiles and birds are closely related.