A previous study in the rat (Pollard et al. 1990) established that caffeine, when administered during pregnancy, significantly inhibited the differentiation of the seminiferous cords and subsequent Leydig cell development in the interstitium. However, that study could not distinguish between the direct effects of caffeine and/or the intermediary secondary toxic effects of metabolites such as theophylline and theobromine. Because the fetus lacks the appropriate enzyme systems, clearance of toxic substances takes place via the placenta and maternal liver. Thus, a suitable in vitro system can effectively differentiate between primary and secondary drug effects. In the present study, 13-day-old fetal testis, at the stage of incipient differentiation, were cultured for 4 days in vitro in the presence of graded doses of caffeine, theophylline or theobromine. It was found that explants exposed to caffeine or theobromine differentiated normally, developing seminiferous cords made up of Sertoli and germ cells, soon followed by the differentiation of functionally active Leydig cells appearing in the newly formed interstitium. However, explants exposed to theophylline failed to develop seminiferous cords and, as a consequence, Leydig cells. In conclusion, insights obtained from different experimental methods, such as organ culture or whole organism studies, are not always identical. It may be prudent, therefore, to take into account that certain experimental techniques, despite providing valuable information, may require confirmation by other test methods in order to obtain an in-depth understanding of mechanisms of action involved.