Somatic cells from the opossums Monodelphis domestica and Didelphis virginiana were labelled with 5-bromodeoxyuridine (BrdU), treated with colchicine, stained with acridine orange and examined using fluorescence microscopy. BrdU-incorporated metaphase spreads from females of M. domestica at developmental stages from late bilaminar blastocysts to adults showed replication asynchrony of the two (acrocentric) X chromosomes. The long arm of one X chromosome was the latest replicating region in the entire chromosome complement and is presumed to represent transcriptional inactivation and X dosage compensation. The minute short arm of the same X, which contains a nucleolar organizer region, was earlier replicating and synchronous with the short arm of its homologue and is thus assumed to escape inactivation. BrdU-incorporated spreads from cells of fetuses, neonates and adults of D. virginiana also showed a late replicating (submetacentric) X chromosome. The pattern was different from that of M. domestica because of the different morphology and the presence of large blocks of constitutive heterochromatin in both homologues. The timing and pattern of replication of the single X in males of both species resembled the earlier replicating X in females. The array of molecular techniques now available offers the best means for investigating X-chromosome replication and activity states of X-linked genes in the earliest stages of marsupial embryogenesis.