Glycerol uptake in Nocardia asteroides exhibited biphasic kinetics suggesting that glycerol was translocated by at least two mechanisms. Uptake of glycerol at concentrations more than 80 μM followed the kinetics of free diffusion, whereas, glycerol concentrations less than 80 μM exhibited saturation kinetics typical of a specific transport system. This latter mechanism had the characteristics of constitutive facilitated diffusion or active transport; however, a definite energy relation was not established. After entry, glycerol was further catabolized by glycerokinase (EC. 2.7.1.30) whose specific activity increased 15-fold when cells were grown in a medium containing glycerol as the sole carbon source. The presence of glycerokinase did not enhance the rate of glycerol transport.