Sperm immobilization factor (SIF) causing 100% immobilization of spermatozoa isolated from Staphylococcus aureus when characterized using LC-MS (Liquid chromatography-mass spectrometry) showed that this 20 kDa protein had peptide sequence similarity with hsp-70 protein. It was found to completely (100%) inhibit Mg(++) ATPase activity of spermatozoa at concentration of 100 μg mL(-1). Sperm samples treated with SIF also showed reduction in calcium ionophore-induced acrosome reaction as compared to control samples (treated with calcium ionophore alone). Binding studies of FITC labelled SIF with spermatozoa using fluorescent microscopy showed binding of SIF to the surface of spermatozoa indicating the presence of SIF binding receptor. The receptor was extracted by 3M NaCl and purified by gel permeation chromatography. Characterization of the receptor by MALDI-TOF (Matrix-assisted laser desorption ionization-time of flight) indicated that the receptor shared sequence similarity with MHC class II antigen. A calorimetric study showed that the receptor moiety on spermatozoa was specific for the purified ligand as binding of the receptor to ligand was enthalpically (-11.9 kJ mole(-1)) as well as entropically (21.53 J mole(-1) K(-1)) favored resulting in the Gibb's free energy of -18.57 kJ mole(-1).