A series of gene duplications that began in a stem species of Mammalia and led to five developmentally regulated hemoglobin (i-chain loci (E, v, a, 6, and (3) in a common ancestor of eutherian orders Artiodactyla, Rodentia, Lagomorpha, and Primates had important consequences in mammalian evolution. Findings reported here indicate that two progenitors of the five linked genes existed by the time of the eutherian (placental mammal)-metatherian (marsupial mammal) split and that these two genes were already differentiated with respect to their promoter regions and developmental expression. Southern blot and sequence analyses of the hemoglobin (i-chain genes of the opossum (Didelphis virginiana) revealed only two genes, one with coding and promoter sequences similar to eutherian prenatally expressed E, y, and iq genes and the other coding for adult opossum hemoglobin (i-chains and having eutherian adult (i-type promoters. The most parsimonious arrangement of >80 (i-globin exon sequences depicts the opossum embryonic-type gene as orthologously related to eutherian E, y, and q genes and the opossum adult-type gene as orthologously related to 6 and (i genes. These data further indicate that after the initial (i duplication in the stem of Mammalia, the locus that became developmentally delayed in its expression evolved at a faster rate than the locus that became embryonically expressed.Previous studies have provided evidence that a series of tandem gene duplications during formative periods of mammalian evolution led to a cluster of five developmentally regulated hemoglobin p-chain loci (5' e-v-71-8-P 3') in which e, y, and q were embryonically expressed genes and 8 and p were adult-expressed genes (1-5). The first of the duplications occurred in the range of 155-200 million years ago (1, 6-8) and produced two gene lines, of which one was the progenitor of E, y, and -q loci and the other was the progenitor of 8 and p3 loci (2-4). The later duplications that separated E, y, and i1 from each other and in parallel 8 and p were estimated to have occurred in the range of 90-140 million years ago (1,(6)(7)(8).The proposed range of dates for the later duplications coincides with the geological period (100-135 million years ago) in which the stem-marsupials and stem-eutherians are thought to have diverged from their last common ancestor (9-11). Clearly, the p-chain-related hemoglobin genes of marsupials need to be examined and compared to those of eutherians to determine if more than two progenitors of E, y, 71, 8, and p loci existed at the time of the metatherian-eutherian split and whether the last common ancestor of marsupials and placental mammals already had p-chainrelated hemoglobin genes that were differentially expressed in embryonic and adult life.