Folate receptor-โฃ (FR-โฃ) is generally over-expressed in non-mucinous human ovarian carcinomas. The meaning of FR-โฃ over-expression and its role in the 5-methyltetrahydrofolic acid (N 5 -CH 3 -H 4 PteGlu) transport in such tumors is not clear, especially compared with the reduced folate carrier (RFC), the other known folate transporter. In this study, we analyzed molecular FR-โฃ and RFC expression in 16 ovarian carcinoma tissues and in 5 ovarian carcinoma cell lines using competitive PCR. Co-expression of the 2 transporters was found both in vivo and in vitro. FR-โฃ mRNA expression in the cell lines was in good agreement with the corresponding protein expression evaluated by cellular folic acid binding and immunofluorescence analysis, using a specific monoclonal antibody (MAb) (MOv18). Moreover, RFC mRNA expression levels were consistent with the selective cellular binding of N-hydroxysuccinimide of [ 3 H]-methotrexate (NHS-MTX). The 5 ovarian carcinoma cell lines (IGROV-1, SW-626, SKOV-3, OVCAR-3 and OAW-42), grown at physiological N 5 -CH 3 -H 4 PteGlu concentrations (20 nM) and expressing FR-โฃ and RFC levels superimposable to those observed in vivo, were used as in vitro cellular model to evaluate the different contribution of FR-โฃ and RFC to the transport of N 5 -CH 3 -H 4 PteGlu. The cytoplasmic N 5 -CH 3 -[ 3 H]H 4 PteGlu accumulation observed in each cell line was approximately linear over 4 hr of incubation, but there was no correlation between the rate of folate internalization and FR-โฃ and RFC expression levels. Furthermore, the selective inhibition of FR-โฃ and RFC functionality allowed us to distinguish their differential role on the overall N 5 -CH 3 -[ 3 H]H 4 PteGlu intracellular delivery. Treatment with the N-hydroxysuccinimide of folic acid, which blocks FR-โฃ activity, showed only a partial inhibition (about 20%) of folate internalization in all the cell lines. In contrast, the inhibition of RFC by NHS-MTX, under conditions that did not affect FR-โฃ functionality, generally reduced folate accumulation by more than 70%. Only one cell line (IGROV-1) showed a comparable contribution of the 2 transport systems. Our findings suggest that in ovarian carcinomas, in spite of its over-expression, FR-โฃ generally plays a minor role in N 5 -CH 3 -H 4 PteGlu transport compared with RFC.