Receptor-dependent four-dimensional quantitative structure–activity relationship (RD-4D-QSAR) studies were applied on a series of 21 peptides reversible inhibitors of Trypanosoma cruzi trypanothione reductase (TR) (Amino Acids, 20, 2001, 145). The RD-4D-QSAR (J Chem Inform Comp Sci, 43, 2003, 1591) approach can evaluate multiple conformations from molecular dynamics simulation and several superposition structure alignments inside a box composed by unitary cubic cells. The descriptors are the occupancy frequency of the atoms types inside the grid cells. We could develop 3D-QSAR models that were highly predictive (q2 above 0.71). The 3D-QSAR models can be visualized as a spatial map of atom types that are important on the comprehension of the ligand–enzyme interaction mechanism, pointing main pharmacophoric groups and TR subsites described in the literature. We were able also to identify some TR subsites for further development in the drug discovery process against tropical diseases not yet studied.