By the use of a newly developed technique of ultrathin-layer electrophoresis, class I and class II alcohol dehydrogenase activity could be demonstrated in microdissected samples of the periportal, intermediate, and perivenous zones of the liver acinus in men and women. It could be demonstrated that both classes exhibit low activity in the periportal zone. From there, a rising gradient in the direction of the perivenous end was apparent. This increase, however, was found to be significant only in women. The analysis of class I alcohol dehydrogenase isoenzymes showed that the expression of alpha-, beta-, and gamma-containing isoforms did not differ in relation to the intraacinar position. The constant proportions of the isoenzymes to the maxima and minima of the total alcohol dehydrogenase activity support the view that the adult liver-specific isoenzyme pattern is determined during postnatal development.