Arachidonic acid (AA) might be released from a number of different intracellular pools. This aspect was studied by sequential incorporation of 3H- and 14C-AA during 24 h in rabbit colonic biopsies in culture. Mucosal explants were subjected to 3H-AA (0-6 h) and 14C-AA (6-24 h) followed by a 2-h washout. Lipid extraction of biopsies revealed a distinct pattern where 3H, in relative terms, dominated in phosphatidylinositol (PI) and phosphatidylethanolamine (PE) and 14C was more abundant in neutral lipids, whereas the remaining lipids had equal proportions of labellings. When stimulating double labelled biopsies with 1 microM phorbol 12-myristate 13-acetate (PMA), 10 micrograms/ml A23187 and a combination thereof, the 14C-labelling was in general released in larger amounts, i.e. a maximum of 7.5% with the combined drugs vs 5% for 3H. However, the relative release of 3H increased upon stimulation especially for PMA + A23187. The release of labelling was directly coupled to a stimulation dependent production of PGE2 in the order PMA less than A23187 less than PMA + A23187, maximum release = 8.2 +/- 2.4 ng/h and 3 biopsies. This study shows the existence of different AA-pools, where PI and PE (3H-labelling) appears to have a slow turnover and representing a terminus in a redistribution chain and where neutral lipids (14C-labelling) have a more rapid turnover. Upon stimulation more AA appears to be recruited from pools with slow turnover.