Non-steady state, ¹³C-labeled volatile fatty acid (VFA) infusions were used to determine the effects of heat stress on ruminal production, absorption, and interconversion rates of VFA. It was hypothesized that increased fluid exit during heat stress would decrease VFA absorption rates. Eight 6-mo-old Holstein heifers were used in a repeated measures experiment with 2 periods and 2 thermal treatments. Heifers were individually housed in thermally controlled rooms and were fed a common high forage ration throughout the experiment. During period 1, both groups of heifers had ad libitum access to feed and were housed at 20°C. During period 2, temperature was increased to 30°C in one room (HS; n = 4) and the heifers kept in the 20°C room (n = 4) were pair-fed so that intake matched HS. Each period consisted of 7 d of adaptation followed by 3 d for determination of VFA kinetics. On each of these days, a bolus of polyethylene glycol (PEG) solution was delivered intraruminally to each heifer at hour 0 to determine fluid volume in the rumen. After an initial priming dose, a constant Co-EDTA infusion was delivered intraruminally over the sampling period to measure fluid exit. A continuous infusion of ¹³C labeled acetate, propionate, or butyrate was initiated at hour 0; VFA infusions were terminated after 6 h and rumen fluid samples were collected at hour 0 and hourly for 12 h. Isotope ratios and concentrations of VFA were measured by gas chromatography, isotope ratio mass spectrometry and gas chromatography, mass spectrometry, respectively, and were used with fluid volume and passage data to parameterize a fully exchanging, dynamic 3 pool model of rumen VFA fluxes. Heat stress increased apparent de novo synthesis of butyrate (P < 0.001); increased fluid-mediated exit of acetate, propionate, and butyrate (P < 0.001, P = 0.002, and P = 0.054, respectively); and decreased absorption of butyrate (P < 0.001). Interconversions among acetate, propionate, and butyrate were not affected by heat stress. The depressed absorption of butyrate, despite the enhanced production rates, could have important post-absorptive implications because butyrate has been shown to help maintain insulin sensitivity.