The flushing of both uterine horns of 36 uterine specimens of buffalo Cows free from any pathological lesions via catheter using 30 ml of phosphate buffer solution (PBS) were collected. Alkaline phosphatase (ALP) and Aspartate aminotransferase (AST) were evaluated. Significant cyclic variations (P < 0.001) of ALP activity was found in uterine flushing media. The ALP activity was significantly higher (P < 0.001) in flushing media obtained from ipsilateral horn to corpus luteum than from contralateral horn. No significant cyclic variations of AST activity was found in uterine flushing media. The results revealed that ALP activity in flushing media of buffalo cows is very low when compared with cattle and causes of this variations were discussed.