The products of short-term (less than 1 min) 14CO2 fixation by A. nidulans were studied. The cells were not in the steady state and thus conclusions can only be drawn about possible pathways. Reproducible patterns could not be obtained. Two types of patterns were observed; in type I fixation 3-phosphoglycerate was the most heavily labeled initial product and in type Ii fixation the most intensely labeled initial product was aspartate. In type Ii patterns the bulk of the label in aspartate was in C4 suggesting a carboxylation of phosphoenolpyruvate. However, 14C incorporation into oxaloacetate was found to be much less than into aspartate. Ribulose-1,5-diphosphate carboxylase activity (sufficient to account for 20% manometric photosynthesis rates) and phosphoenolpyruvate carboxylase activity (sufficient to account for whole-cell rates of synthesis of aspartate) were observed in cell-free extracts. However, a phosphoenolpyruvate-stimulated carboxylation of ribulose diphosphate was not detected indicating that the label in aspartate was not a reflection of a pathway involving transcarboxylation of oxaloacetic acid. Early label detected in glutamate-1-14C probably arose from oxaloacetate-4-14C via the citric acid cycle rather than by the reductive carboxylation of succinic acid as α-ketoglutarate synthetase activity was not detected in extracts. In conclusion the Calvin cycle was the major route of CO2 fixation but a second, minor path of CO2 incorporation was the carboxylation of phosphoenolpyruvate.