The effect of a homologous series of aliphatic n-alkanols on the presence of water within the head group and acyl chain region of lipid bilayers was investigated using time-resolved fluorescence spectroscopy according to a previously published approach [Ho, C., Slater, S. J., & Stubbs, C. D. (1995) Biochemistry 34, 6188-6195]. Upon addition of n-alkanols to phosphatidylcholine bilayers the fluorescence lifetime of N-[5-(dimethylamino)naphthalene-1-sulfonyl]dipalmitoylphosphatidyleth anolamine (dansyl-PE) decreased, indicative of an increased water content within the head group region, the effect being a linear function of n-alkanol chain length (C1-C8), based on the total n-alkanol concentration. The fluorescence lifetimes of 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3, 5-hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-phosphatidylcholine (DPH-PC) and N-[[4-(6-phenyl-1,3, 5-hexatrienyl)phenyl]propyl]trimethylammonium p-toluenesulfonate (TMAP-DPH), and the fluorescence intensity ratio of the latter in D2O compared to that in H2O, were used to probe the level of water in the acyl chain region. There was a decrease in the lifetime and an increase in the D2O/H2O fluorescence intensity ratio upon addition of short-chain n-alkanols (C1-C3), suggestive of increased water content. By contrast, long-chain n-alkanols (C4-C8) increased the lifetime and decreased the ratio, suggestive of decreased water content. Acyl chain order, determined from DPH-PC fluorescence anisotropy, was decreased by all n-alkanols, indicating that the effects were not probe-dependent. The effects of short- and long-chain n-alkanols on the fluorescence lifetime of the tryptophans of gramicidin, incorporated into phosphatidylcholine bilayers as a model membrane protein, were similar to those obtained with TMAP-DPH and DPH-PC; ethanol decreased and hexanol increased the lifetime. Thus the effect of n-alkanols and general anesthetics on changes in the amount of water that may be accommodated within the acyl chain region of the bilayer is not predictable on the basis of the magnitude of effects on head group region or acyl chain order/fluidity.