Lactobacillus salivarius cells contain an inducible transport system for folate. Influx via this system is time- and temperature-dependent, requires glucose and glutamine for optimum activity, and is half-maximal at folate concentrations in the nanomolar range. The folate internalized after 30 min at 30 degrees C is not released from the cells by excess extracellular folate and is recovered in cell extracts primarily in metabolized forms. A membrane-associated folate-binding protein is also present in cells that have been induced to transport folate. This binding protein constitutes 1% of total cellular protein, exhibits a high affinity for folate (KD = 0.40 nM), and requires divalent cations for optimum binding activity. Folate binds rapidly to this protein, while the exchange of bound substrate with folate added subsequently is relatively slow and dependent on the metabolic state of the cell. The transport rate per binding site is 0.05/min at 30 degrees C. A comparison of substrate specificity showed that folate binding and transport are both inhibited to the same extent by several different folate compounds, and a parallel irreversible inhibition of both processes is observed by prior treatment of the cells with a carbodiimide-activated derivative of folic acid. Binding protein labeled covalently with [3H]folate and solubilized with Triton X-100 was purified by a fractionation procedure involving absorption and elution from microgranular silica and molecular sieve chromatography. The isolated protein appeared homogeneous by gel electrophoresis and had an apparent molecular weight of 21,000. Monoclonal antibodies to the folate transport protein of Lactobacillus casei showed a high degree of cross-reactivity to the isolated binding protein from L. salivarius, indicating that these proteins share common epitopes. These results suggest that folate uptake by L. salivarius proceeds via an abundant membrane-associated binding protein which facilitates the movement of folate across the membrane as an electroneutral complex with cations. The substrate then slowly dissociates from internalized binding sites and is metabolized sequentially to coenzyme forms and then to membrane-impermeable folylpolyglutamates.