We evaluated the prevalence and characteristics associated with hyperhomocysteinemia in ankylosing spondylitis (AS). Ninety-seven patients with AS were compared with 97 controls. The assessment included clinical characteristics, disease activity (BASDAI), functioning (BASFI), history of drugs, and erythrocyte sedimentation rate (ESR). Total serum homocysteine (tHcy) was determined by fluorescence polarization immunoassay. A higher frequency of hyperhomocysteinemia (>15 micromol/L) was observed in AS (12 vs. 1%, P = 0.002). In the multivariate analysis the risk for hyperhomocysteinemia was increased in patients with higher score of HAQ-S (OR = 5.27, 95% CI: 1.29-21.44) and higher ESR (OR = 1.09, 95% CI: 1.02-1.18). No statistical associations was observed between hyperhomocysteinemia with other variables including methotrexate or sulfasalazine utilization. In conclusion, this study found a significant prevalence of hyperhomocysteinemia in Mexican patients with AS mainly associated to a worst functional impairment. Further follow-up studies are required to evaluate the risk of cardiovascular disease in these patients.