Abstract: This study was undertaken to evaluate the viability and infectivity of the tachyzoites in different temperatures and times of milk by bioassay methods. Tachyzoites of RH strain were transferred into sterile milk and kept in different temperatures (4, 20 and 37°C) for different time intervals (10 and 30 min) and their viability was evaluated by vital stain (methylene blue) and the infectivity by intra-peritoneal and oral inoculation to different groups of inbred BALB/C mice (2-4 mice in each group obtained from Pasteur Institute, Tehran, Iran). Tachyzoites in saline were intra-peritoneally inoculated to inbred BALB/C mice as positive controls. All animals were kept in Laboratory Animal Center of Shiraz University of Medical Sciences in Shiraz, southern Iran. The touch smear of spleen and liver of dead mice were stained by Giemsa and were parasitologically evaluated. Eighty percent of tachyzoites were stained by methylene blue indicating identical parasite viability. All mice died after intra-preritoneal inoculation of tachyzoites present in sterile milk. Only one out of eight mice which was orally inoculated remained alive. All mice fed by tachyzoites in saline died. Duration of mice vitality was more in oral route than peritoneal inoculation (n1 = 11, Mean1 = 9.55 days and n2 = 17, Mean2 = 6.65 days, p = 0.001). Tachyzoites in drinks and foods undergoing heating up to 37°C for 30 min such as milk can be a possible route of transmission of toxoplasmosis to human. Therefore strict public health measures should be carried out to prevent transmission of disease in high risk areas.