In a previous study on monovular cows, follicles revealed a mean antral (follicular fluid) temperature 1.54°C cooler than rectal temperatures in ovulating cows, whereas no such temperature differences were detected in non-ovulating cows. The present study adds to our previous work, this time considering 24 bi-ovular cows (one follicle per ovary). In order to increase the number of pre-ovulatory follicles failing to ovulate, this study was performed under heat-stress conditions. Follicular temperatures of the ovulating follicles (n = 31) were 0.93°C significantly cooler (P < 0.0001) than rectal temperatures, whereas no significant differences in temperature were found in non-ovulating follicles (n = 17). Eight cows became pregnant. The results of the present study indicate that, similar to those in monovular cows, pre-ovulatory follicles in bi-ovular cows were cooler than deep rectal temperatures and those temperature gradients were not found in follicles showing ovulation failure.