The relationship between exercise performance and lactate and ventilatory thresholds under two distinct environmental conditions is unknown. We examined the relationships between six lactate threshold methods (blood- and ventilation-based) and exercise performance in cyclists in hot and cool environments. Twelve cyclists performed a lactate threshold test, a maximal O(2) uptake (Vo(2max)) test, and a 1-h time trial in hot (38°C) and cool (13°C) conditions, before and after heat acclimation. Eight control subjects completed the same tests before and after 10 days of identical exercise in a cool environment. The highest correlations were observed with the blood-based lactate indexes, however, even the indirect ventilation-based indexes were well correlated with mean power during the time trial. Averaged bias was 15.4 ± 3.6 W higher for the ventilation- than the blood-based measures (P < 0.05). The bias of blood-based measures in the hot condition was increased: the time trial was overestimated by 37.7 ± 3.6 W compared with only 24.1 ± 3.2 W in the cool condition (P < 0.05). Acclimation had no effect on the bias of the blood-based indexes (P = 0.51) but exacerbated the overestimation by some ventilation-based indexes by an additional 34.5 ± 14.1 W (P < 0.05). Blood-based methods to determine lactate threshold show less bias and smaller variance than ventilation-based methods when predicting time-trial performance in cool environments. Of the blood-based methods, the inflection point between steady-state lactate and rising lactate (INFL) was the best method to predict time-trial performance. Lastly, in the hot condition, ventilation-based predictions are less accurate after heat acclimation, while blood-based predictions remain valid in both environments after heat acclimation.