Pemetrexed (PMX) is a 5-substituted pyrrolo[2,3-d]pyrimidine antifolate used for therapy of nonsquamous nonsmall cell lung cancer (NS-NSCLC). PMX is transported by the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). Unlike RFC, PCFT is active at acidic pH levels characterizing the tumor microenvironment. By real-Time reverse-Transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, PCFT transcripts and proteins were detected in primary NS-NSCLC specimens. In six NS-NSCLC cell lines (A549, H1437, H460, H1299, H1650, and H2030), PCFT transcripts and proteins were detected by real-Time RT-PCR and western blots, respectively. 6-Substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates related to PMX [compound 1 (C1) and compound 2 (C2), respectively] are selective substrates for PCFT over RFC. In the NS-NSCLC cell lines, both [3H]PMX and [3H]C2 were transported by PCFT. C1 and C2 inhibited proliferation of the NS-NSCLC cell lines; A549, H460, and H2030 cells were more sensitive to C1 than to PMX. C1 and C2 inhibited glycinamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis. When treated at pH 6.8, which favors PCFT uptake, C1 and C2 inhibited clonogenicity of H460 cells greater than PMX; PMX inhibited clonogenicity more than C1 or C2 at pH 7.2, which favors RFC transport over PCFT. Knockdown of PCFT in H460 cells resulted in decreased [3H]PMX and [3H]C2 transport and decreased growth inhibition by C1 and C2, and to a lesser extent by PMX. In vivo efficacy of C1 was seen toward H460 tumor xenografts in severe-combined immunodeficient mice. Our results suggest that 6-substituted pyrrolo[2,3-d] pyrimidine thienoyl antifolates offer significant promise for treating NS-NSCLC by selective uptake by PCFT.