We measured intra- and extravascular body-fluid compartments in 12 resting males before (day 1; control), during (day 8) and after (day 22) a three-week, exercise-heat acclimation protocol to investigate plasma volume (PV) changes. Our specific focus was upon the selective nature of the acclimation-induced PV expansion, and the possibility this expansion could be sustained during prolonged acclimation. Acclimation was induced by cycling in the heat, and involved sixteen treatment days (controlled hyperthermia (90 min); core temperature = 38.5°C) and three experimental exposures (40 min rest, 96.9 [SD 9.5] min cycling), each preceded by a rest day. The environmental conditions were: 39.8°C [SD 0.5], relative humidity 59.2% [SD 0.8]. On days 8 and 22, PV was expanded and maintained relative to control values (day1: 44.0 ±1.8, day 8: 48.8 ±1.7 and day 22: 48.8 ±2.0 ml·kg^-1; P<0.05). The extracellular fluid compartment (ECF) was equivalently expanded from control values on days 8 (279.6 ±14.2 versus 318.6 ±14.3 ml·kg^-1; n=8; P<0.05) and 22 (287.5 ±10.6 versus 308.4 ±14.8 ml·kg^-1; n=12; P<0.05). Plasma electrolyte, total protein and albumin concentrations were unaltered following heat acclimation (P>0.05), although the total plasma content of these constituents was elevated (P<0.05). The PV and interstitial fluid (ISF) compartments exhibited similar relative expansions on days 8 (15.0 ±2.2% versus 14.7 ±4.1%; P>0.05) and 22 (14.4 ±3.6% versus 6.4 ±2.2%; P=0.10). It is concluded that the acclimation-induced PV expansion can be maintained following prolonged heat acclimation. In addition, this PV expansion was not selective, but represented a ubiquitous expansion of the extracellular compartment.