Studied is the effect of the non-ionic surfactant Tween 80 on the microbial transformation of 4-androstene-3,17-dione into its 9a-hydroxy-derivative by resting Rhodococcus sp. cells. The surfactant was applied in the cultivation medium as an additional source of carbon, in the transformation reaction medium as a mediator of the steroid substrate solubility or was used for permeabilization of the glucose grown Rhodococcus sp. cells. Special attention is paid to the fact that Tween 80 accelerates the 9a-steroid hydroxylation reaction carried out by glucosegrown cells. When the surfactant was applied as a supplementary source of carbon, the rate of the steroid hydroxylation reaction was significantly lower. In addition, the kinetics of the transformation process changed into a linear one thus indicating a very slow, if any, product degradation. The fatty acid profile, cell surface hydrophobicity as well as cell ultrastructure observed by scanning and transmission electron microscopy in the Tween 80-and glucose-grown Rhodococcus sp. cells are compared and related with their 9a-hydroxylating activity.