The potent nonpolyglutamatable dihydrofolate reductase inhibitor N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-o rnithine (PT523) and six of its B-ring (5-deaza, 8-deaza, and 5,8-dideaza) analogues were compared in terms of their ability to: (a) inhibit the growth of CCRF-CEM human leukemic lymphoblasts, and (b) utilize the reduced folate carrier (RFC) in these cells as measured in a competition assay of [(3)H]methotrexate ([(3)H]MTX) influx. The IC(50) values of the hemiphthaloylornithine derivatives against CCRF-CEM cells after 72 hr of drug exposure varied from 0.64 to 1.3 nM as compared with 14 nM for MTX and 4.4 nM for aminopterin (AMT). The K(i) values of these compounds in the [(3)H]MTX influx assay were in the 0.3 to 0.7 microM range as compared with a K(i) of 5.4 microM for AMT and a K(t) of 7.1 microM for MTX. As a group, the affinities of these compounds for the RFC were approximately 10-fold greater than those of their respective glutamate analogues. These results indicate that, in addition to their previously reported tight binding to dihydrofolate reductase, a property contributing to the high potency of PT523 and its B-ring analogs as inhibitors of tumor cell growth is their strong affinity for the RFC.