The present study was undertaken to elucidate the effect of non-luteal oviductal proteins on sperm characteristics in Murrah buffaloes. Oviducts from healthy buffaloes were collected immediately after slaughter and the oestrous cycle phase was determined as either luteal or non-luteal based on ovarian morphology. Non-luteal oviducts (n = 80) were flushed from the isthmic end of the oviduct with PBS, fluid was centrifuged at 10,000 g at 4 degrees C for 20 min and then dialysed and clarified. The supernatant obtained was lyophilized to concentrate the protein and stored at -20 degrees C till use. Sixteen good quality ejaculates from four Murrah buffalo bulls were collected using an artificial vagina. After fresh semen analysis, each ejaculate was split into two parts and extended in Tris-citrate-egg yolk glycerol dilutor. Part I of the split ejaculate was treated with non-luteal oviductal proteins at the dose rate of 1 mg/ml of diluted semen, while part II remained as control. The extended semen was equilibrated for 4 h at 5 degrees C, filled in 0.5 ml French straws, exposed to LN(2) vapour, plunged into LN(2) and then stored at -196 degrees C. The equilibrated and frozen-thawed semen was evaluated for sperm motility, viability, acrosomal integrity, cervical mucus penetration test and hypo-osmotic sperm swelling test (HOST). In frozen-thawed semen, the percentage of sperm motility, viability and acrosomal integrity was significantly (p < 0.05) higher in the treatment group compared to the control group. The incorporation of non-luteal oviductal proteins in the extender increased the ability of sperm to penetrate cervical mucus both after equilibration and the freeze-thaw process. Similarly, the proportion of sperm with intact plasma membrane, as revealed by HOST values, was also significantly (p < 0.05) higher in the treatment group (32.6%) than the control group (27%) in frozen-thawed semen. It was inferred that incorporation of non-luteal whole oviductal fluid proteins improved the sperm quality in frozen-thawed semen in Murrah buffaloes.