Hemoglobin (Hb) genes have been cloned from three different species of ciliated protists, P. multimicronucleatum, P. triaurelia and P. jenningsi. Southern blotting of the genomic DNAs using the P. caudatum Hb cDNA showed both intraspecies variation in different stocks of P. caudatum and interspecies variation within the genus Paramecium. The isolated Hb genes were composed of 118, 117 and 117 codons, and interrupted by a short intron with 27, 29 and 29 bp at the same position, in P. multimicronucleatum, P. triaurelia and P. jenningsi, respectively. This suggests that the one-intron and two-exon structure has been conserved in the Hb genes in this genus. The amino acid sequences of the Paramecium Hbs were more than 87% identical to one another and homologous to those from the other ciliated protists Tetrahymena thermophila and T. pyriformis, the green alga Chlamydomonas eugametos, and the cyanobacterium Nostoc commune Hbs, all of which consist of about 120 amino acid residues (120-aa group). In particular, the amino acid sequences of the P. triaurelia and P. jenningsi Hbs were the same, although there were 20 nucleotide differences between the coding regions in the two genes. A maximum likelihood inference as to the phylogenetic relationships among these genes suggests that the Paramecium Hbs genes have evolved more rapidly than the other genes in the 120-aa group, and that P. triaurelia and P. genningsi are sibling species and the P. aurelia complex became a small cell after it separated from P. jenningsi.