OBJECTIVES: To determine the association between the α-actinin-3 (ACTN3) R577X polymorphism and elite team-sport athletic status in three cohorts of European team-sport athletes. DESIGN: We compared the genotype and allele frequencies of the ACTN3 R577X (rs1815739) polymorphisms between team-sport athletes (n=205), endurance athletes (n=305), sprint/power athletes (n=378), and non-athletic controls (n=568) from Poland, Russia and Spain; all participants were unrelated European men. METHODS: Genomic DNA was extracted from either buccal epithelium or peripheral blood using a standard protocol. Genotyping was performed using several methods, and the results were replicated following recent recommendations for genotype-phenotype association studies. RESULTS: Genotype distributions of all control and athletic groups met Hardy-Weinberg equilibrium (all p>0.05). Team-sport athletes were less likely to have the 577RR genotype compared to the 577XX genotype than sprint/power athletes [odds ratio: 0.58, 95% confidence interval: 0.34-0.39, p=0.045]. However, the ACTN3 R577X polymorphism was not associated with team-sports athletic status, compared to endurance athletes and non-athletic controls. Furthermore, no association was observed for any of the genotypes with respect to the level of competition (elite vs. national level). CONCLUSIONS: The ACTN3 R577X polymorphism was not associated with team-sport athletic status, compared to endurance athletes and non-athletic controls, and the observation that the 577RR genotype is overrepresented in power/sprint athletes compared with team-sport athletes needs to be confirmed in future studies.