Folylpolyglutamate synthetase (FPGS) catalyzes the sequential addition of several glutamates to folate, forming gamma-linked polyglutamate folates of varying lengths. To understand how this protein is capable of accommodating ligands of different length and net charge, we have performed docking studies for folate substrates and glutamate based on the ternary crystal structure of Lactobacillus casei FPGS. Our results suggest two locations for folate binding, the one seen in the crystal structure and another distinct cavity. According to our model and experimental data, it is highly probable that folate can bind in both sites, and we suggest that the new pocket is especially important for the initial addition of the first glutamate residue. Docking longer substrates, di- and triglutamylated folates, showed how these molecules bind in the same sites. The longer folates also adopted transition-state-like conformations that may help us to understand the ligation reaction in FPGS and influence the design of mechanism-based inhibitors for anticancer or antimicrobial therapy.