Six healthy Thoroughbred mares were trained to run on a high-speed treadmill and were conditioned for approximately 5 weeks. Each horse performed 6 identical standardised exercise tests (SET) 5 to 7 days apart Each SET was performed under different ambient laboratory conditions: low temperature/low humidity (LL) 20.2° ± 0.6°C, 53.6 ± 4.1%; low temperature/high humidity (LH) 19.7 ± 0.6°C, 86.7 ± 4.2%; medium temperature/low humidity (ML) 24.6 ± 0.2°C, 58.7 ± 3.7%; medium temperature/high humidity (MH) 24.7 ± 0.3°C, 87.5 ± 1.4%; high temperature/low humidity (HL) 31.1 ±0.6°C, 41.6 ± 3.5%; and high temperature/high humidity (HH) 30.6 ± 0.2°C, 84.4 ± 3.6%. Horses completed the 46 min SET, became fatigued or exercise was terminated when a pulmonary artery temperature (PAT) of 41.5°C was reached. During a 30 min recovery period, horses stood quietly on the treadmill. Only one trial was terminated due to fatigue; in all other trials horses completed the 46 min SET or the trial was terminated when PAT=41.5°C. The relationships among run time (min) or PAT (°C), ambient temperature and % relative humidity were defined by multiple linear regression analysis. Run time (min) = 90.9 — (1.39 × ambient temperature °C) — (0.236 × relative humidity %). Approximately 54% of the variation in run times was explained by variation in environmental conditions, indicating that ambient temperature and humidity contribute substantially to the rate of rise in PAT. Run times to a PAT of 41.5°C were significantly shorter (P<.000001) in HH than in other groups during the SET. Approximately 63% of the variation in PAT at the end of exercise was due to ambient temperature and humidity. In general, the longer the exercise time, the better the correlation among PAT, ambient temperature and relative humidity. Rectal temperature increased significantly (P<.0001) during the recovery period only in horses in the HH group, indicating that heat dissipation during recovery may have been impaired by environmental conditions. This study demonstrates that hot humid conditions result in more rapid rate of rise in PAT for horses undertaking identical exercise tests.