We have used atomistic molecular dynamics simulations to consider 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescent probes in a fluid dipalmitoylphosphatidylcholine bilayer with 5 and 20 mol % cholesterol (CHOL). We show that while DPH affects a number of membrane properties, the perturbations induced by DPH depend on the concentration of cholesterol in a membrane. For example, we find DPH to influence the mass density distribution of lipids across the membrane and to promote the ordering of acyl chains around the probe. Yet, these perturbations get relatively weaker for increasing cholesterol concentration. Meanwhile, we also find that the commonly used analysis in terms of the Brownian rotational diffusion (BRD) model with Legendre polynomials to interpret fluorescence anisotropy (FA) experiments is sensitive to the amount of cholesterol. For small concentrations of cholesterol, the analysis of FA turns out to yield a relatively good approximation of the correct orientational distribution of DPH. However, for a CHOL concentration of 20 mol %, we find that the FA analysis fails to yield the true orientational distribution of DPH, the disagreement being substantial. The results suggest that in highly ordered membrane domains, the view given by FA analysis using the BRD model is likely reliable in a qualitative sense, but the quantitative description deviates substantially from the correct one. The present results imply that FA studies for the orientational distribution of DPH should be interpreted with great care.