The binding of deoxynucleoside 5'-monophosphates and various folate derivatives to Lactobacillus casei thymidylate synthetase was measured by equilibrium dialysis. The substrate, deoxyuridylate (dUMP), and the product, thymidylate (dTMP), were bound to the enzyme at a ratio of unity and appeared to compete for the same site. The binding of each was tighter in 50 mM Tris-HCl (pH 7.1) than in 50 mM potassium phosphate (pH 7.0). Folate derivatives increased the affinity of the enzyme for the substrate to a greater extent than for the product, although they themselves did not appear to be bound in the absence of substrate or substrate analogues. However, in the presence of enzyme, dUMP or 4-N-OH-dCMP, and either 7,8-dihydrofolate or methotrexate, a ternary complex was obtained, with the folate derivatives exhibiting single site binding. The binding of dUMP in the ternary complex was 25-fold greater than that of 7,8-dihydrofolate and 50-fold greater than that of methotrexate. Supporting evidence for the enhanced stability of the ternary complex was provided by heat inactivation studies. As in the case of deoxynucleotide binding to the synthetase, the ternary complex was more stable in Tris HCl than in potassium phosphate buffer. The binding characteristics of the substrate analogue 5-fluoro-2'-deoxyuridylate (FdUMP) could be clearly distinguished from that of dUMP by comparing their binding in phphate and Tris-HCl. While each deoxynucleotide exhibited only single site binding in phosphate, a second site was clearly demonstrated for FdUMP with Tris-HCl. The binding of FdUMP to each site appeared to be equal in the presence of methotrexate or (-)5,10-methylene tetrahydrofolate and was increased about 17-fold in Tris-HCl. Two sites were also obtained for FdUMP in the presence of 7,8-dihydrofolate, but Scatchard analyses revealed a biphasic curve, with the second site possessing a higher dissociation constant than the first. A second low affinity FdUMP binding site was also detected in phosphate buffer when 7,8-dihydrofolate or (-)5,10-methylene tetrahydrofolate was included in the binding assay. In the presence of (+)5,10-methylene tetrahydrofolate, however, 2 mol of FdUMP was bound stoichoimetrically to 1 mol of enzyme regardless of the buffer used. The significance of these results is discussed in relation to the presence of two apparently identical subunits in the native enzyme.