Tumor-targeted specificities of 6-substituted pyrrolo[2,3- d]pyrimidine analogues of 1, where the phenyl side-chain is replaced by 3',6' (5, 8), 2',5' (6, 9), and 2',6' (7, 10) pyridyls, were analyzed. Proliferation inhibition of isogenic Chinese hamster ovary (CHO) cells expressing folate receptors (FRs) α and β were in rank order, 6 > 9 > 5 > 7 > 8, with 10 showing no activity, and 6 > 9 > 5 > 8, with 10 and 7 being inactive, respectively. Antiproliferative effects toward FRα- and FRβ-expressing cells were reflected in competitive binding with [3H]folic acid. Only compound 6 was active against proton-coupled folate receptor (PCFT)-expressing CHO cells (∼4-fold more potent than 1) and inhibited [3H]methotrexate uptake by PCFT. In KB and IGROV1 tumor cells, 6 showed <1 nM IC50, ∼2-3-fold more potent than 1. Compound 6 inhibited glycinamide ribonucleotide formyltransferase in de novo purine biosynthesis and showed potent in vivo efficacy toward subcutaneous IGROV1 tumor xenografts in SCID mice.