The average membrane location of a series of diphenylhexatriene (DPH)-derived membrane probes was analyzed by measuring the quenching of DPH fluorescence with a series of nitroxide-labeled lipids in which the depth of the nitroxide group is varied. All DPH derivatives were located deeply within the bilayer. Some derivatives were anchored at a shallower depth than free DPH by attachment to cationic or anionic groups. However, the absolute change in DPH depth upon attachment to such groups was relatively modest (<4 A). In fact, protonated DPH fatty acid and a DPH fatty acyl group attached to a phosphatidylcholine were found to locate slightly more deeply than free DPH. The location of DPH derivatives can be explained by the length of the DPH group and its tendency to orient predominantly parallel to the fatty acyl chains of the bilayer. These factors allow a charged group attached to one end of a DPH molecule to be accommodated at the polar surface while maintaining a deep DPH location. Basically, it appears that most DPH derivatives probe the same region in the bilayer. We conclude previously reported differences in fluorescence polarization of free and anchored forms of DPH may reflect a direct effect of anchoring on motion rather than an effect on average DPH location. Other experiments showed the localization of DPH probes was found to be similar in the presence and absence of cholesterol. This implies that previously observed cholesterol-induced effects on DPH fluorescence polarization also largely reflect differences in DPH motion, not DPH location. From the quenching results it was also possible to define rules governing the location of a variety of chemical groups in membranes by comparison of the results obtained with DPH derivatives to those of similar derivatives of other fluorescent groups. Finally, an important goal of this study was to compare different methods of analysis of quenching data: parallax analysis, distribution (Gaussian) analysis (using a single Gaussian), and a second-order polynomial analysis. To evaluate the accuracy of these methods, the apparent depths of a series of fluorescence probes previously analyzed by parallax analysis was reanalyzed with all three methods. There was good agreement unless the fluorescent molecule was very shallow or very deep. In such cases, only parallax analysis gave physically reasonable results. This is likely to be due to the lack of a sufficient number of quenchers spanning a wide enough range for other analyses to compensate for deviations from ideal curves. Parallax analysis was also compared to distribution (Gaussian) analysis using a double Gaussian fit to account for quenching from the trans leaflet (Ladokhin, A. (1997) Methods Enzymol. 278, 462-473). Again more physically reasonable results were obtained from parallax analysis, likely due to non-Gaussian behavior of the depth dependence of quenching. Notwithstanding these observations, the significant number of cases where Gaussian curve fitting methods for quenching analysis are most powerful are discussed.