The alpha-isoform of the glycosylphosphatidylinositol cell membrane tethered folate receptor (alpha-FR) is overexpressed in some carcinomas (notably ovarian carcinomas) relative to normal tissues. The nonpolyglutamatable folate-based thymidylate synthase (TS) inhibitor, CB300638 (TS K(i) = 0.24 nM) displayed an IC(50) of 0.0028 microM for the inhibition of the growth of human A431-FBP cells transfected with the alpha-FR. In contrast, the IC(50) for the neotransfected A431 cells was 0.81 microM (300-fold higher). Similarly, this compound inhibited the growth of human KB cells that constitutively overexpress the alpha-FR with an IC(50) of 0.0036 microM. These data were derived from cells grown in a physiological concentration of folate (20 nM R,S-leucovorin). Incubation of KB cells with a 1 microM excess of folic acid (FA), to selectively block uptake via the alpha-FR, increased the CB300638 IC(50) to 0.39 microM. The relatively low potency of CB300638 under these conditions, or in cell lines not expressing the alpha-FR, is ascribed to its low affinity for the ubiquitously expressed folate transporter, the reduced-folate carrier (K(i) for inhibition of [(3)H]methotrexate transport >100 microM). The high potency of CB300638 in alpha-FR-overexpressing cell lines is attributable to high affinity of the alpha-FR (53% of FA) and efficient endosomal trafficking mediated by the alpha-FR. Sixteen-h exposure to CB300638 inhibited the rate of (3)H(2)O release from 5-[(3)H]dUrd (in situ TS assay) in A431, A431-FBP, and KB cells with IC(50) values of 0.1 microM, 0.005 microM, and 0.002 microM, respectively. The coaddition of 1 micro M FA increased the IC(50)s for A431-FBP and KB cells to approximately 0.1 microM consistent with alpha-FR-mediated transport of CB300638. In conclusion, alpha-FR-mediated uptake of CB300638 leads to TS and growth inhibition that is highly selective for alpha-FR overexpressing tumor cell lines. The low expression of the alpha-FR in normal tissues, particularly those sensitive to TS inhibitors, together with the low affinity of CB300638 for the reduced-folate carrier, suggests that the compound may have potential as an antitumor agent with a high therapeutic index.