The protherian mammals consist of three species: the platypus, the Australian echidna and the Niugini echidna. These mammals diverged from the therian line of descent about 150-200 million years ago; hence comparisons of gene arrangements and gene control mechanisms between prototherian and therian mammals may yield significant data about gene rearrangements during mammalian evolution and about the evolution of complex genetic control systems. The chromosome complements of the three monotreme species are highly conserved. In particular, the X (or X1) chromosomes are G-band identical and share considerable G-band homology with the Y chromosomes. Replication asynchrony between X chromosomes suggests that X chromosome inactivation operates in females, and is apparently tissue- specific (as it is in marsupials), and confined to the differential region of the X (X1) chromosome (as it is in eutherian mammals). These results suggest that sex chromosome differentiation in the monotremes represents an intermediate stage in the evolution of the dimorphic sex chromosomes of therian mammals and that X-chromosome inactivation may also represent a comparatively primitive stage. Studies of gene location in the platypus using platypus-rodent cell hybrids suggested that HPRT and PGK are syntenic in the platypus, but it was not possible to assign the syntenic group to a particular chromosome. In situ hybridisation was used to assign three genes, located on the X in eutherians and marsupials, to the monotreme X. However, human X short-arm markers were found by in situ hybridisation to be autosomal in monotremes (as they are in marsupials). A model for the evolution of mammalian sex chromosome differentiation and X-chromosome inactivation is presented in which a gradual reduction of the Y chromosome, and recruitment of newly unpaired loci on the X into a system of X-chromosome inactivation, has accompanied eutherian evolution.