This study determined exposure of pregnant women to ochratoxin A (OTA). Forty samples of first-void urine samples from Croatian women in the third trimester of pregnancy were analyzed for OTA and its major metabolite ochratoxin alpha (OTα). The subjects filled a short food frequency questionnaire (FFQ). Analysis was performed by HPLC-FLD following liquid-liquid extraction. All samples were subjected in parallel to enzymatic treatment (β-glucuronidase/aryl sulfatase) to release OTA and OTα from the conjugates. The median urinary levels of OTA and OTα before treatment were 0.02 (range: nd-1.07) ng/mL and 0.16 (nd-1.86) ng/mL; the concentrations after enzyme hydrolysis were 0.02 (nd-1.11) ng/mL and 1.18 (0.11-7.57) ng/mL. While OTα levels increased significantly following enzymatic treatment, evidence for OTA conjugation was weak. The ratio of urinary OTα medians after and before hydrolysis was 1.5 times higher than previously reported for nonpregnant female subjects, possibly indicating upregulated metabolism and/or elimination of the mycotoxin and metabolites in pregnancy. The mean daily dietary OTA intake calculated from FFQs (1.08±0.57 ng/kg body weight) was well below the provisional tolerable daily intake and the greatest contributors to intake were cereal products, fruit juices, chocolate and coffee.