A novel single-step microbial transformation process for the production of testosterone from cholestrol by Lactobacillus bulgaricus in an aerated fermenter was investigated. The metabolism of glucose possibly supplying reducing power as NADH was necessary for the reduction of androst-4-en-3,17-dione (AD) to testosterone (TS). The growth period for the accumulation of testosterone in maximal amount and the residual glucose increased in parallel with the amount of glucose supplement in growth cultures. After the glucose in the fermentation culture was completely exhausted, most of the testosterone was oxidized to AD. Addition of a larger amount of glucose could prevent oxidation of testosterone to AD. The biotransformation of cholestrol was significantly increased in the presence of cyclodextrin (CD) in the fermenting medium. The addition of 0.1% CD to the growth medium facilitated the transport of the steroid substrate through the microbial cell wall.