Uptake of [14C]glycerol by 72-h-old resting cell suspensions of Tieghemiomyces parasiticus was linear with respect to time and had the characteristics of a protein-mediated system of transport. The uptake process obeyed Michaelis–Menten kinetics with an apparent Km of 8 × 10−4 M and Vmax of 5.26 nmol ∙ min−1 ∙ mg−1 dry wt. Glycerol uptake exhibited pH and temperature dependence with optima at pH 5 and 35 °C, respectively. The energy of activation (Ea) and Q10 for this uptake process were calculated to be 12 043 cal/mol and 1.98, respectively, between 20 and 30 °C. Glycerol uptake also exhibited substrate specificity and was inhibited by α-glycerolphosphate and 1,2-propanediol, while inhibition by 1,3-propanediol was negligible. Preexposing resting cell suspensions to sodium azide and 2,4-dinitrophenol (4 × 10−3 M) significantly inhibited glycerol uptake, possibly suggesting an energy requirement for transport. Cold osmotic shock treatment severely reduced glycerol uptake but did not destroy cell viability.