(With Plate i and 4 figures in the text)In the main body of the present paper we shall report and discuss the results of physiological experiments performed during the period 1947-9 to strengthen the evidence that COj-fixation is a metabolic event invariably associated with and related to dark acidification in plants showing Crassulacean acid metabolism. We shall view these results against a general background of modern biochemical knowledge, but we shall not give more than incidental consideration to details of partial reactions.We have confirmed (see § II) all the results obtained in 1946 which were reported for Kalanchoe (see p. 2) and Bryophyllum, briefly by and more fully by Thomas & Beevers (1949). Further (see § III), we have extended our investigations to certain other genera in the list Bennet-Clark (1933a) gave of plants showing diurnal variation in acidity. For most of the species clear evidence was obtained of CO2 absorption in the dark from atmospheres enriched in carbon dioxide. Fluctuations in values of apparent respiratory quotients in such atmospheres, and in ordinary air, can be related to stages in Crassulacean acid metabolism in the dark (Thomas, 1949;Wolf, 1949). Accordingly, in most of our experiments we have measured oxygen uptake as well as CO2 output (positive or negative) and the accumulation or disappearance of titratable acid. In general, during dark acidification either in C02-enriched atmospheres or in air, fluctuations in R.Q.'s have appeared to relate to the extent of CO2-fixation and acid accumulation over the period of measurement.To obtain further evidence that acid accumulation and CO2-fixation march together, experiments were performed at different temperatures ( § IV) and in atmospheres containing different concentrations of carbon dioxide ( § V). The results obtained were in accord with the view ( § II, (i)) that the enzyme bringing about CO^-fixation is an integral part of the mechanism which occasions the conversion of carbohydrate into malic acid.Throughout we assume that over the periods of our experiments, changes in titratable acidity relate predominantly to the production and consumption of malic acid (see p. 6), summarized by overall equations (ii) and (iii) respectively. From equation (ii) we have calculated the malic acid equivalent of the amount of CO2 fixed, i.e. the malic acid production according to our hypothesis. In § VI we discuss possible reasons why, in our experiments, malic acid accumulation, as measured by increase in titratable acidity, has usually been less than the calculated value of malic acid production. New Phytol. 53, i '