Two experiments were designed to assess the potential utility of the propionate challenge test (PCT) as an index of gluconeogenic capacity. In Expt. 1, the dose-response to jugular propionate infusion was assessed in a duplicated 4 x 4 Latin square experiment with 8 lactating dairy cows. Sodium propionate (4.5 mol/L, pH 7.4) was infused in an intrajugular bolus at 0 (saline), 0.52, 1.04, or 1.56 mmol/kg body weight (BW), and jugular blood was sampled over the following 2 h. Peak propionate concentration in plasma and area under the curve for plasma glucose both increased linearly with increasing propionate dose (P < 0.01). Plasma free fatty acid (FFA) concentration was elevated by all propionate treatments at 20 min postinfusion (P = 0.03), and plasma cortisol concentration tended to increase (P < 0.10) after propionate infusions. Experiment 2 was designed to study the effect of short-term differences in fed state on responses to propionate infusion. Lactating dairy cows (n = 8) were included in a duplicated 4 x 4 Latin square design with a 2 x 2 factorial arrangement of treatments. Sodium propionate (1.04 mmol/kg BW) or saline was infused either before feeding (0900) or 2 h after feeding (1300). Fed cows consumed 4.4 +/- 1.4 kg dry matter before the PCT. Although fed cows had a significantly higher preinfusion plasma propionate concentration, fed state did not influence postinfusion changes in plasma propionate, glucose, insulin, glucagon, or FFA concentrations. Liver glycogen concentration decreased significantly after propionate, but not saline infusion (P < 0.05). Short-term differences in fed state do not affect the physiological responses to PCT. However, glucagon release after jugular administration of propionate is likely supraphysiologic, and postinfusion lipolysis and glycogenolysis suggest that stress responses may alter PCT measurements. Although the PCT may help to diagnose liver dysfunction, it is not a useful index with which to assess differences in gluconeogenic capacity.