A relationship between soil respiration rate (R_s) and temperature (T_s), has been understood to be predicated on carbon (C) substrate availability. However, unlike T_s, C availability in soils is not a state variable that can be readily measured. The C in soils has come from plants, so the C supply rate can be affected by the weather and nutrient supply. We studied a fertile soil beneath pasture, measuring R_s across a temperate – climate range of T_s. Our objectives were to: (1) quantify the synchrony of diurnal changes in T_s and R_s beneath pasture under conditions favourable for plants, (2) quantify responses of microbial respiration (R_m) to the removal of plants and depletion of C supply over time at various T_s, and (3) determine if R_m was related to water-soluble (WSC, 20°C) and hot-water-soluble C (HWSC, 80°C) contents. At a grassland site, R_s increased with T_s as predicted by an Arrhenius type relationship. Sampled soil was incubated at 3°, 9°, and 24°C and R_m was measured over 14 days. In addition soil samples were pre-incubated at 3° or 9°C for both 5 and 14 days, then incubated at 24°C for 1 day and R_m was measured. On day 2, R_m was less than predicted at 24° and 9°C, respectively, suggesting a C availability limitation. The time courses of R_m, revealed that at 24°C, R_m utilised C that was not utilised at lower T_s, indicating that evidently recalcitrant C was available to microbes at a warmer temperature. The responses of R_m at 24°C after the pre-incubation treatments were identical for the 3°C and 9°C pre-incubation treatments, although significantly more C was respired during pre-incubation at 9°C. The WSC and HWSC contents were unaffected by T_s, so did not provide useful measures of the C substrate available for R_m.