The inhibitory effect of heat treatment on CO₂ fixation in isolated chloroplasts was increased as the medium pH was lowered. At pH 8.1 heat treatment at 35℃ for 10 min produced a 50% inhibition while the same treatment caused a nearly 90% inhibition at pH 6.0. As a result, involvement of stromal acidification as an intermediate step in the heat-induced inhibition of CO₂ fixation was suggested. Addition of alkalinizing agents (NH₄Cl and KCl) to the medium prior to heat treatment of chloroplasts did not prevent inhibition. Also, addition of these agents to heated chloroplasts did not reverse or alleviate inhibition, suggesting stromal acidification is not involved. Consequently, direct measurement of stromal pH was attempted. Heat treatment did not lower the stromal pH either at pH 8.1 or at pH 6.0. Rather, it slightly raised the stromal pH. Thus, stromal acidification as an intermediate step for the heat induced inhibition of CO₂ fixation in isolated chloroplasts can be excluded. Reconstituted chloroplasts revealed thylakoidal damage after heat treatment at pH 6.0, which did not occur at pH 8.1. It seems that this additional damage in thylakoids is the cause for the higher degree of inhibition at pH 6.0.