Acid extract of mature sperm of the toad, Bufo japonicus, exclusively comprised sperm-specific basic proteins which moved faster than somatic histones on acid/urea/Triton X-100 polyacrylamide gel electrophoresis. When these proteins were purified by reversed-phase high-performance liquid chromatography they were found to consist of three components; one of these was a phosphorylated form of another, so that there were only two distinct components (P1 and P2). Amino acid sequence analyses indicated that the components both contained 39 amino acid residues, with 43.6% Arg, and differed only in the 28th amino acid residue (P1, Asp; P2, Glu). They had molecular masses of 5092 Da (P1) and 5106 Da (P2). The nucleotide sequences of cDNA clones encoding P1 (245 bases) and P2 (305 bases) showed that the difference in the amino acid residue between P1 and P2 was due to the difference of a nucleotide at position +87. Both cDNAs possessed a canonical signal (AATAAA) for polyadenylation and/or cleavage of transcript at the 3' untranslational region. Statistical analyses of amino acid sequence similarities suggested that the Bufo protamines are homologous with the protamines of fishes rather than with those of avian/mammalians.