CD4(+) T follicular helper (T-FH) cells are central for generation of long-term B-cell immunity. A defining phenotypic attribute of T-FH cells is the expression of the chemokine R CXCR5, and T-FH cells are typically identified by co-expression of CXCR5 together with other markers such as PD-1, ICOS, and Bcl-6. Herein, we report high-level expression of the nutrient transporter folate R 4 (FR4) on T-FH cells in acute viral infection. Distinct from the expression profile of conventional T-FH markers, FR4 was highly expressed by naive CD4(+) T cells, was downregulated after activation and subsequently re-expressed on T-FH cells. Furthermore, FR4 expression was maintained, albeit at lower levels, on memory T-FH cells. Comparative gene expression profiling of FR4(hi) versus FR4(lo) Ag-specific CD4(+) effector T cells revealed a molecular signature consistent with T-FH and T(H)1 subsets, respectively. Interestingly, genes involved in the purine metabolic pathway, including the ecto-enzyme CD73, were enriched in T-FH cells compared with T(H)1 cells, and phenotypic analysis confirmed expression of CD73 on T-FH cells. As there is now considerable interest in developing vaccines that would induce optimal T-FH cell responses, the identification of two novel cell surface markers should be useful in characterization and identification of T-FH cells following vaccination and infection.