The main objective was to evaluate the effects of seasonal variations in air temperature, soil water content, and irradiation on the net rate of CO₂ uptake for Agave tequilana growing in a warm subtropical environment (Amatitan, Jalisco, Mexico) and in a temperate subtropical environment (Arandas, Jalisco, Mexico). At Amatitan, the lowest value of positive daily net CO₂ uptake (141 mmol/m²/d) occurred at the end of the summer rainy season and the highest values during the winter (578 and 921 mmol/m²/d); at Arandas, the lowest value (188 mmol/m²/d) occurred at midsummer and the highest values at the end of the summer (763 mmol/m²/d) and in late winter (572 mmol/m²/d). The highest values of daily net CO₂ uptake in both localities reflected prolonged daily periods of net CO₂ uptake that included both daytime and nighttime assimilation accompanied by high rates of nocturnal net CO₂ uptake due to moderate nighttime air temperatures. High temperatures in the summer reduced daily net CO₂ uptake at both localities but in late summer, the nighttime temperatures at Arandas were moderate, which lead to high daily net CO₂ uptake. Leaf succulence allowed substantial daily net CO₂ uptake when the soil water content was relatively low during the winter and the spring (January–March). Indeed, appreciable daily net CO₂ uptake occurred throughout the year for A. tequilana at both localities, indicating that this CAM plant species can sequester carbon even during prolonged dry periods.