Colonic mucosal biopsies cultured for 6 h in the presence of cycloheximide (CH) showed a dose-dependent inhibition of protein synthesis but a biphasic PGE2 production pattern with an increase in both basal and A23187 stimulated PGE2 release at 0.2 microM. At 10 microM CH both protein synthesis as well as basal and PMA induced PGE2 production was inhibited by 90% whereas A23187 stimulated release showed a 50% decrease. At a dose of 100 microM, CH totally blocked also A23187 stimulated PGE2 release without much further decrease in protein synthesis. The effects of 10 microM CH were time-dependently reversible. In biopsies loaded with 3H-arachidonic acid (AA), 10 microM CH had no apparent effect on phospholipase A2 activity, nor could exogenous AA overcome the CH inhibition of basal PGE2 release. No inhibition of prostaglandin synthetase (PS) activity was found in homogenates of biopsies treated with 10 microM CH for 6 h. No direct effect of CH (up to 1 mM) was seen in control homogenates. It is concluded that at least one step in the PGE2 production is protein synthesis dependent. The effect is however not due to a limitation in the enzymes of the major PS system but more likely to one of its co-factors. This factor only plays a role in the intact cell and its importance seems to be reduced during A23187 conditions possibly due to altered cell status and/or other sources of PS. Commonly used high doses (100 microM) of CH give unspecific effects unrelated to inhibition of protein synthesis.