We have examined the phase partition preferences of the even chain length (n = 10-22) diacyl-3'3'-indocarbo-cyanine iodides (Cn diI) incorporated in disaturated lecithin (PC) vesicles. Two parameters were used to determine this phase preference: (i) the direction of shift of the phase transition temperature (Tm) induced by the dyes and (ii) the self-quenching of fluorescence due to aggregation in the gel phase of those dyes which preferentially partition into the fluid. Dyes that lower Tm preferentially partition into the fluid phase; those that raise Tm preferentially partition into the gel. By these criteria in dimyristoyl-PC, C10 diI and C12 diI preferentially partition into the fluid phase, C14 diI and C16 diI show no preferential partition, C18 diI preferentially partitions into the gel, and C20 diI and C22 diI preferentially partition into the fluid. In dipalmitoyl-PC, the pattern of preference is identical with that observed in dimyristoyl-PC, only shifted to longer chain length diI's by two carbons. Diffusion measurements by fluorescence photobleaching recovery of these dyes in gel-phase multilayers showed tham all to be immobile, D less than 10(-10) cm2/s, while in fluid-phase multilayers thay all had diffusion coefficients of D approximately 10(-8) cm2/s independent of chain length. In mixed-phase multilayers, however, each Cn diI showed mobile fraction which reflected its phase-partition preference.