Systematic examinations were made of factors influencing bacteriorhodopsin formation during the growth of Halobacterium halobium. Light-induced adenosine triphosphate (ATP) production and [14C]proline uptake were used as measures of functional ability of the purple membrane. Maximum bacteriorhodopsin formation occurred under growth conditions of illumination and limited aeration. The purple membrane -ATP production system did not confer an appreciable growth advantage. Growth in the dark or with adequate aeration partially suppressed bacteriorhodopsin formation and the effects were additive. Nicotine effectively inhibited bacteriorhodopsin formation. A rapid synthesis of functional pigment occurred when washed suspensions of cells which had been grown under illumination with nicotine present were incubated under dark, aerobic conditions. The alleviation of this nicotine inhibition was not blocked by chloramphenicol or bacitracin. Bacteriorhodopsin formation was negligible when washed suspensions of cells from dark, limited aeration or light, adequate aeration cultures were incubated in the light with limited aeration. A nutritionally complex medium was needed to elicit appreciable bacteriorhodopsin formation by the cells from the dark or adequately aerated cultures. Bacitracin partially inhibited this bacteriorhodopsin formation by cells form the light, adequately aerated culture.