trans-Parinaric acid, a polyene fatty acid, is widely used as a fluorescent membrane probe in photophysical and biophysical studies. We have worked on the orientational order of this molecule in Langmuir-Blodgett multilayer films of both gel-phase dipalmitoylphosphatidylcholine and arachidic acid at room temperature. Namely, orientational density probability functions were calculated using the maximum entropy method. Because liquid-crystal lipids do not deposit forming multilayers by Langmuir-Blodgett techniques, we have used Brownian dynamics methods to simulate a trans-parinaric acid molecule in a liquid-crystal bilayer environment. Orientational density probability functions are also achieved under the ergodic hypothesis. In the gel phase, trans-parinaric acid is highly ordered (narrow density probability function) and almost perpendicular (78 degrees) to the multilayer planes. In the liquid-crystal phase, the density probability function broadens and the average orientation is not so close to the multilayer normal axis. The highly ordered system detected in the gel phase is in agreement with the preference for the gel phase in the partitioning of this probe among different phases, which is a very peculiar behavior. If its inclusion was not a favorable process, i.e., not adjusted to the membrane geometry, it could not have such a strict alignment. The change in orientational distribution upon phase transition is in agreement with published data on the dynamics of the probe in the nanosecond time range.