Recent studies demonstrate that nitric oxide synthase (NOS) is involved in sweating during intermittent exercise in the heat in young adults (Stapleton, Exp Physiol 99:921, 2014; Fujii, J Physiol 2014, in press). However, it remains unknown which NOS isoform mediates NOS‐dependent sweating. We tested the hypothesis that endothelial NOS (eNOS), but not inducible NOS (iNOS) or neuronal NOS (nNOS), contributes to NOS‐dependent sweating during intermittent exercise in the heat. Seven young (24 ± 4 years), healthy adults (4 males, 3 females) peformed two 30‐min bouts of cycling at a fixed rate of metabolic heat production (400 W; equivalent to ~50 %VO2max) in the heat (35 ºC). Exercise was separated by a 20‐ and 40‐min recovery respectively. Forearm sweat rate (ventilated capsule) was evaluated at four skin sites instrumented with intradermal microdialysis fibres that were continuously perfused with: 1) lactated Ringer (Control), 2) 5mM Nω‐amino‐L‐arginine (LNAA, eNOS inhibitor), 3) 0.1mM N‐3‐aminomethyl‐benzyl‐acetamidine (1400W, iNOS inhibitor), or 4) 5mM Nω‐propyl‐L‐arginine (NPLA, nNOS inhibitor). Forearm sweat rate (mg min‐1 cm‐2) during the last 5 min of the first exercise was reduced with 1400W (0.72 ± 0.04, P = 0.035) but not LNAA (0.83 ± 0.05, P = 0.550) or NPLA (0.84 ± 0.05, P = 0.878) in comparison to Control (0.90 ± 0.08). A similar pattern of response was observed throughout the second exercise bout. We show that iNOS, but not nNOS or eNOS, mediates NOS‐dependent sweating during moderate intensity intermittent exercise in the heat in young adults. Support: Natural Sciences and Engineering Research Council of Canada (RGPIN‐06313‐2014).