Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is a functional candidate gene with susceptibility to rheumatoid arthritis (RA). The aim of this work was to investigate the possible influence of the recently described CT60A/G dimorphism of the CTLA-4 gene in the susceptibility to RA in Spanish patients. A total of 433 RA patients and 398 control subjects were included in the study. Genotyping of CTLA-4 CT60 was performed using two different methods: polymerase chain reaction restriction fragment length polymorphism system using an amplification-created restriction site and a TaqMan 5'-allelic discrimination assay. In order to validate results obtained by different methods, a quality-control exercise was performed. No significant deviation in the distribution of the alleles or genotypes of the CT60 was found when we compared RA patient and control groups. In addition, no differences in CTLA-4 CT60 genotypic distribution was found when RA patients and controls were stratified by the presence or absence of the shared epitope. In conclusion, our results do not support an association between CT60A/G polymorphism and susceptibility to RA in the Spanish population, although the contribution of other positions located within the 3' region of the CTLA-4 gene to RA susceptibility cannot be discarded.