Short-term exposure to elevated levels of free fatty acids (FFA) perturbs a variety of cellular functions. It is frequently observed that cis-unsaturated fatty acids (FA) mediate these perturbations while trans-unsaturated or saturated FA are relatively inert. This dichotomy has generally been ascribed to the differential effects of these FA on membrane structure, specifically, that cis but not saturated FA alter lipid acyl chain order. Direct support for this view, however, is lacking because membrane partition of FA has not been determined for the relevant FA and for the conditions of the functional studies. Previous measurements of membrane partition of natural long-chain FA have relied on the determination of the amount bound to the membrane rather than the aqueous-phase concentration of FA. Because [FFA] is low, however, the partition coefficient (Kp) is relatively insensitive to the membrane-bound concentration and, therefore, accurate determinations of Kp require direct [FFA] measurements. In this study FA partition between the aqueous phase and either lipid bilayer vesicles or cytotoxic T lymphocytes (CTL) was measured using a recently developed fluorescent indicator of FFA. This indicator is composed of recombinant intestinal fatty acid binding protein derivatized with acrylodan and is termed ADIFAB. Using ADIFAB, partition coefficients were determined for seven saturated and cis-unsaturated FA under conditions that parallel those in which cis FA have been shown to inhibit CTL signaling. In general, Kp values were approximately an order of magnitude greater than previous determinations and were found to be greater for the noninhibitory saturated FA than the inhibitory cis FA.(ABSTRACT TRUNCATED AT 250 WORDS)