Pathways of glucose catabolism, potentially operational in six strains of obligately aerobic, acidophilic bacteria, including Acidiphilium cryptum strain Lhet2, were investigated by short-term radiorespirometry and enzyme assays. Short-term radiorespirometry was conducted at pH 3.0 with specifically labeled [C]glucose. The high rate and yield of C-1 oxidized to CO(2) indicated that the Entner-Doudoroff, pentose phosphate, or both pathways were operational in all strains. Apparent nonequivalent yields of CO(2) from C-1 and estimated CO(2) from C-4 (C-1 > C-4) were suggestive of simultaneous glucose catabolism by both pathways in all strains tested. Variation in the relative contribution of the two pathways of glucose catabolism appears to account for observed strain differences. Calculation of the actual percent pathway participation was not feasible. Enzyme assays were completed with crude extracts of glucose-grown cells to substantiate the results obtained by radiorespirometry. The key enzymes of the pentose phosphate pathway (6-phosphogluconate dehydrogenase) and the Entner-Doudoroff pathway (2-keto-3-deoxy-6-phosphogluconate aldolase and 6-phosphogluconate dehydrase) were present in all strains examined (PW2, Lhet2, KLB, OP, and QBP). However, none of the strains exhibited detectable levels of the key enzyme of the Embden-Meyerhof-Parnas pathway, 6-phosphofructokinase. All strains contained glucose-6-phosphate dehydrogenase and fructose bisphosphate aldolase. The results of the enzyme study supported the contention that the pentose phosphate and Entner-Doudoroff pathways are operational for glucose catabolism in the acidophilic heterotrophs, and that the Embden-Meyerhof-Parnas pathway is apparently absent.