The STATs are a family of transcription factors. STAT5A, previously known as MGF, transduces prolactin signals to the milk protein genes. Here, we describe the detection of nucleotide sequence polymorphism in exon 16 of the bovine STAT5A gene, coding for the SH2 domain. SSCP was found in a 281-bp PCR amplified gene fragment, lying between positions 12,525 and 12,806, and encompassing parts of intron 15 and exon 16 of the bovine STAT5A gene (GenBank AJ 237937). Three SSCP patterns (genotypes) were identified in a group of 108 animals of different cattle breeds. The DNA sequencing showed that they differed by a CCT deletion at position from 12,549 in intron 15, and a T-->C substitution at position 12,743 in exon 16. The latter mutation changes an amino acid sequence in the STAT5A protein - a Val/Ala substitution at position 686. Since T-->C substitution creates a new MslI site, genetic variants in the bovine STAT5A gene can be distinguished with RFLP analysis. The frequency of alleles T and C varied between the different cattle breeds studied; the CC genotype was the least frequent and the frequency of alleles T and C was 0.842 and 0.158, respectively. Proteins were extracted from the cell nuclei of liver tissues derived from bulls of different STAT5A genotypes and subjected to EMSA in order to study if the amino acid substitution might change the DNA-binding capacity of STAT5A transcription factor. Statistically significant (p<0.05) differences in nuclear protein binding to DNA were observed between genotypes TT and CC; nuclear proteins derived from CC animals always showed less DNA protein complexing than those of TT animals. EMSA competition experiments confirmed that STAT5 transcription factors take part in the formation of the DNA-protein complexes.