We present the results of a comparative differential calorimetric and Fourier transform infrared spectroscopic study of the effect of cholesterol and five analogues on the thermotropic phase behaviour and organization of dipalmitoylphosphatidylcholine bilayer membranes. These sterols/steroids differ in both the nature and stereochemistry of the polar head group at C3 (ßOH, αOH or C=O) and in the position(s) of the double bond(s). In the Δ(5) sterols/steroid series, the concentration of these compounds required to abolish the DPPC pretransition, inversely related to their relative ability to disorder gel state DPPC bilayers, decreases markedly in the order ßOH>αOH>C=O. However, in the Δ(4,6) series, these concentrations are similar, regardless of polar head group chemical structure. Similarly, the residual enthalpy of the DPPC main phase transition at 50mol% sterol/steroid, which is inversely related to the miscibility of these compounds in the DPPC bilayer, also increases in the order ßOH>αOH>C=O, but this effect is attenuated in the Δ(4,6) series. In the two pairs of sterol epimers, the Δ(4,6) compounds exhibit a greater decrease in the temperature and enthalpy of both the pretransition and the main phase transition, whereas the opposite result is observed in the ketosteroid pair. Similarly, the ability of these compounds to order the DPPC hydrocarbon chains decreases in the order ßOH>αOH>C=O in both series of compounds, but in the two pairs of sterol epimers, hydrocarbon chain ordering is greater for the Δ(5) than the Δ(4,6) sterols, whereas the opposite is the case for the steroid pair. Thus, the characteristic effects of sterols/steroids on fluid lipid bilayers are optimal when an OH group rather than C=O group is present at C3, and when this OH group is in the equatorial orientation. We suggest that the presence of keto-enol tautomerism in the conjugated Δ(4,6) ketosteroid may provide additional H-bonding opportunities to adjacent DPPC molecules in the bilayer, which results in more cholesterol-like effects.