Channel and nonchannel forms of gramicidin A (GA) were studied by ESR in various lipid environments using new mono- and double-spin-labeled compounds. For GA channels, we demonstrate here how pulse dipolar ESR can be used to determine the orientation of the membrane-traversing molecule relative to the membrane normal and to study subtle effects of lipid environment on the interspin distance in the spin-labeled gramicidin channel. To study nonchannel forms of gramicidin, pulse dipolar ESR was used first to determine interspin distances corresponding to monomers and double-helical dimers of spin-labeled GA molecules in the organic solvents trifluoroethanol and octanol. The same distances were then observed in membranes. Since detection of nonchannel forms in the membrane is complicated by aggregation, we suppressed any dipolar spectra from intermolecular interspin distances arising from the aggregates by using double-labeled GA in a mixture with excess unlabeled GA. In hydrophobic mismatching lipids (L(β) phase of DPPC), gramicidin channels dissociate into free monomers. The backbone structure of the monomeric form is similar to a monomeric unit of the channel dimer. In addition to channels and monomers, the double-helical conformation of gramicidin is present in some membrane environments. In the gel phase of saturated phosphatidylcholines, the fraction of double helices increases in the following order: DLPC < DMPC < DSPC < DPPC. The equilibrium DHD/monomer ratio in DPPC was determined. In membranes, the double-helical form is present only in aggregates. In addition, we studied the effect of N-terminal substitution in the GA molecule upon channel formation. This work demonstrates how pulsed dipolar ESR may be utilized to study complex equilibria of peptides in membranes.