Myostatin (GDF-8) is a key protein responsible for skeletal muscle growth and development so mutations in the mstn gene can have major economic and breeding consequences. The aim of the present study was to investigate polymorphism in the 5’flanking region of the mstn gene and its possible influence on the myostatin level in skeletal muscles of Polish Black-and-White bulls. The relation between expression of myostatin and another member of the TGF-β superfamily, TGF-β₁, was also examined. We uncovered polymorphism in the 5’flanking region of the mstn gene: G/C substitution at position -7828 (relative to translation start codon ATG). The most frequent genotype was GC (43.6%), followed by genotypes CC (34.2%) and GG (22.2%). The concentration of the active form of myostatin (26 kDa) in M. semitendinosus of homozygotes (CC) was the lowest, whereas the expression of this cytokine in heterozygotes was the highest. The changes in mstn expression did not, however, influence average carcass traits (weight of valuable cuts and weight of lean in valuable cuts). The pattern of differences in the TGF-β₁ concentration corresponded to that observed for myostatin.