In order to study controls on metabolic processes in soils, we determined the dynamics of ¹³CO₂ production from two position-specific ¹³C-labeled pyruvate isotopologues in the presence and absence of glucose, succinate, pine, and legume leaf litter, and under anaerobic conditions. We also compared ¹³CO₂ production in soils along a semiarid substrate age gradient in Arizona. We observed that the C from the carboxyl group (C₁) of pyruvate was lost as CO₂ much faster than its other C atoms (C_2,3). Addition of glucose, pine and legume leaf litter reduced the ratio between ¹³CO₂ production from 1-¹³C pyruvate and 2,3-¹³C pyruvate (C₁/C_2,3 ratio), whereas anaerobic conditions increased this ratio. Young volcanic soils exhibited a lower C₁/C_2,3 ratio than older volcanic soils. We interpret a low C₁/C_2,3 ratio as an indication of increased Krebs cycle activity in response to carbon inputs, while the higher ratio implies a reduced Krebs cycle activity in response to anaerobic conditions. Succinate, a gluconeogenic substrate, reduced ¹³CO₂ production from pyruvate to near zero, likely reflecting increased carbohydrate biosynthesis from Krebs cycle intermediates. The difference in ¹³CO₂ production rate from pyruvate isotopologues disappeared 4–5 days after pyruvate addition, indicating that C positions were scrambled by ongoing soil microbial transformations. This work demonstrates that metabolic tracers such as pyruvate can be used to determine qualitative aspects of C flux patterns through metabolic pathways of soil microbial communities. Understanding the controls over metabolic processes in soil may improve our understanding of soil C cycling processes.