Data are presented on the characterization of two nucleotide sequences found exclusively in the DNA of male dogs. In polymerase chain reactions (PCRs) of canine genomic DNA with a decanucleotide primer of arbitrary sequence (OP-W17), two nucleotide segments (650 and 990 bp) were amplified only from male samples, whereas a number of other fragments between 400 and 2500 bp in size were amplified from both male and female samples. The two malespecific segments were cloned and sequenced, and terminal 24mer oligonucleotide primer pairs were synthesized. PCR with these specific primer pairs resulted in amplification of the two male-specific sequences only from DNA samples of 34 male dogs; no product was amplified from 42 samples of females. A segment of the SRY gene previously localized on the Y chromosome could be amplified in DNA samples that had the two new sequences. EcoRI digested genomic male DNA when hybridized with the 650 bp or the 990 bp sequences, resulted in a single band for each on Southern analysis; DNA from females did not yield any bands. Comparisons between the two new sequences and the SRY gene segment revealed no homologies. We concluded that the two new sequences are specific for the canine Y chromosome and do not contain the short characterized segment of the SRY gene.