Hemoglobin (Hb) purified from the water flea, Daphnia magna, reared under hypoxia was analyzed by two-dimensional gel electrophoresis. The Hb was shown to be composed of six major subunit chain species (designated as DHbA to DHbF). The NH2-terminal amino acid sequences of DHbA, DHbB, DHbC, and DHbF are different from one another, indicating that at least four Hb genes are present in D. magna. The NH2-terminal amino acid sequences of DHbD and DHbE are the same as those of DHbA and DHbB, respectively. The six Hb chains were also found in the animal reared under normoxia in small amounts and with altered composition; the extent of decrease under normoxia was higher in the amounts of DHbC, DHbD, and DHbF than those of others. These results indicate that the Hb genes are differentially regulated by the ambient oxygen concentration. Four Hb genes constituting a cluster in the order, dhb4, dhb3, dhb1, and dhb2, were found on the chromosome of D. magna. The complete nucleotide sequences of the dhb1, dhb2, and dhb3 genes and their cDNAs showed that the genes have a seven-exon, six-intron structure. The structure consists of an intron separating an exon encoding a secretory signal sequence, two large repeated regions of a three-exon, two-intron structure that encode each a domain containing a heme-binding site, and an intron bridging the two repeated regions. The deduced amino acid sequences of the gene products showed higher than 79% identity to one another and showed unique features conserved in D. magna Hb chains. The analysis also suggested that DHbB (or DHbE), DHbF, and DHbC are encoded by the dhb1, dhb2, and dhb3 genes, respectively.