The major role of the high-affinity folate-binding protein (FBP) is to regulate cellular folate homeostasis by increasing folate uptake in case of extracellular and intracellular folate deficiency. On this basis, we hypothesised that the overexpression of FBP in ovarian carcinoma might be physiologically associated with folate deficiency in the extracellular fluids, where ovarian carcinoma cells develop in vivo, or it might be the result of a reduced intracellular regeneration of the 5-methyltetrahydrofolate (5-CH3H4 folate). To test these hypotheses, we determined the bioavailability of folate in serum and in ascitic/cystic fluids of ovarian carcinoma patients (n = 36). The intracellular shortage of 5-CH3H4 folate was evaluated in the extracellular fluids by measuring the concentration of homocysteine (Hcy), which is a useful marker of intracellular folate deficiency. Patients with ascites from malignant and benign non-ovarian pathologies were used as controls (n = 30). We found no folate shortage in the serum and ascitic/cystic fluids of ovarian carcinoma patients. The folate concentration was within the normal range and superimposable on that observed in serum and ascites of control patients. However, the ascitic/cystic Hcy concentration was significantly higher (P < 0.005) than the corresponding serum concentration in a large fraction of ovarian carcinoma patients (72%, 26/36), whereas it was higher only in a small fraction of patients with non-ovarian malignant ascites (24%, 4/17), and in no patient with benign ascites. Hcy accumulation in ovarian carcinoma patients was not associated with a defect in the catabolic pathway of Hcy to cysteine, but was consistent with an impaired remethylation process of Hcy to methionine caused by an intracellular shortage of 5-CH3H4 folate. This suggests a possible association between FBP overexpression and a biochemical defect of the cellular folate metabolism involved in the methionine cycle.