The assembly of the 16-kDa polypeptide channel units in membranes from hepatopancreas of Nephrops norvegicus has been studied both by electron microscopy and by the lipid-protein interactions reported with spin-labeled lipids. Membranes prepared by extraction with detergent have a low lipid/protein ratio (ca. 4-6.5 phopholipids and 1 cholesterol per 16-kDa monomer), and those prepared by alkaline extraction have a higher lipid/protein ratio (ca. 12-16 phospholipids and 3.5-4 cholesterol per 16-kDa monomer). The electron spin resonance (ESR) spectra from lipids spin-labeled at the C-14 position of the (sn-2) chain show lower mobility for the membranes extracted with detergent than for the alkaline-extracted membanes. At higher temperatures, the ESR spectra reveal a population of lipids whose mobility is restricted by direct interaction with the intramembranous sections of the channel assemblies. The population of protein-associated spin-labeled phosphatidylcholine in the alkali-extracted membranes corresponds to 4-5 phospholipid molecules plus 1 cholesterol molecule per 16-kDa polypeptide monomer. These numbers are only slightly smaller than the number of lipid molecules that can be accommodated around the perimeter of the model proposed for the channel complex that consists of a hexameric arrangement of transmembrane four-helix bundles (figure shows Fourier projection map of negatively stained ordered 16-kDa protein-containing membranes extracted with detergent together with lipid molecules). The 16-kDa polypeptide displays a selectivity relative to phosphatidylcholine for negatively charged spin-labeled lipids, stearic acid, phosphatidylserine, and phosphatidylglycerol, suggesting that basic amino acid residues are located close to the lipid headgroups in the channel assembly.