1. Slices or bits of rabbit tissues, not exceeding 100 mg, were incubated in tissue culture medium containing tritium-labelled prostaglandin ([(3)H]PG). In some experiments, incubation medium also contained saturating concentrations of an unlabelled prostaglandin (PG), or [(14)C]-sucrose for determination of extracellular space. At the end of the incubation period, usually 1 hr, the tissues were removed and weighed, and their (3)H (and (14)C) content were determined along with that of a unit volume of medium.2. Tissues known to play a central role in PG metabolism (lung and liver) and in its excretion (kidney cortex) and tissues which have a known function in blood-brain and blood-ocular barriers (choroid plexuses and ciliary processes) show a large accumulation of (3)H when incubated in a medium containing [(3)H]PGE(1). In addition, tissues of the female reproductive tract, and the aorta of the rabbit show similar (3)H accumulation. When uncorrected for tissue solid content or extracellular water volume, the extent of this accumulation is two- to sixfold. Calculated on the basis that all excess (3)H is present in the free form in the intracellular water, the accumulation ratio for ciliary processes, for example, indicates an over fortysix-fold gradient of PGE(1) across the cell membrane.3. Tissues which accumulate [(3)H]PGE(1) also accumulate [(3)H]PGA(1), [(3)H]PGF(1alpha) and [(3)H]PGF(2alpha). In some tissues specificity is, however, apparent; in the lung accumulation of [(3)H]PGA(1) was significantly greater than that of [(3)H]PGF(1alpha).4. The extent of [(3)H]PGE(1) accumulation was decreased, or in some tissues completely inhibited, by incubation at 2 degrees C, or by addition of large concentrations of unlabelled PG.5. Accumulation of [(3)H]PGE(1) by the foetal liver is not apparent on the 20th day of gestation, but is fully developed by the 30th day of gestation. The foetal lung does not accumulate [(3)H]PGE(1) at any stage of gestation.6. In some tissues, most notably muscle, there appears to be full equilibrium of [(3)H]PGE(1) between tissue water and medium within 1 hr of incubation.7. PGs are partially excluded from the intracellular volume of some other tissues, most notably the spleen and subcutaneous connective tissues. This apparent exclusion cannot be blocked by incubation in the cold, or by the addition of saturating levels of unlabelled PG.8. The simplest explanation for all observed results is that cell membranes are, in general, impermeable to PGs. However, there are specific, carrier-mediated mechanisms across some membranes which facilitate the entry of PGs. In some cells these transport mechanisms are linked to a source of metabolic energy, and/or to the counter-transport of some other substance, thus allowing net accumulation of PGs against a concentration gradient. Alternatively, (3)H accumulation may represent adsorption of [(3)H]PGs or one of their labelled metabolites on to specific adsorption sites.