The rates of leakage of CO2 from plant assimilation chambers must be known in order to correct measurements of net photosynthesis and respiration for this unwanted but omnipresent source of error. Similarly, in order to precisely calculate heating or cooling requirements for plant growth structures, air leakage or “infiltration” must be known. The objective of this paper is to derive theoretically exact equations for calculating such leakage rates from measurements of CO2 concentration [CO2]. The needed equations were derived from the solution of a basic differential CO2 balance equation. The procedure presumes the determination will be done when plant CO2 exchange is steady, a condition most nearly satisfied by respiration near the end of the night. The equation had two unknowns, respiration and leakage rate. From measurements before and after a CO2 perturbation, it was possible to derive sets of two equations with two unknowns, which were then solved for the respiration and leakage rates. In contrast to prior work, the new equations are theoretically exact, and they can account for changes in ambient [CO2] during the measurement periods and also for differing rates of CO2 injection before and after the perturbation. Moreover, these theoretically exact equations are no more complex than the prior approximate equations for a couple of practical cases. Calculations for three specific cases showed the approximation used in the prior equations introduced errors of 0.8, 5.5, and 1.6%. Thus, using these equations derived in this paper should permit more accurate calculations of leakage rate and ultimately, therefore, more accurate determinations of gas exchange rates from plants in assimilation chambers as well as of the heating or cooling requirements of the chambers themselves.