A choline-requiring strain of mouse fibroblast cells (LM cells) was cultured in suspension with choline, N,N'-dimethylethanolamine, N-monomethylethanolamine, or ethanolamine. These choline analogues were incorporated into membrane phospholipids as phosphatidyl-N,N'-dimethylethanolamine, phosphatidyl-N-monomethylethanolamine, and phosphatidylethanolamine. Plasma membranes, microsomes, mitochondria, and their respective lipids were isolated and the characteristic temperatures were determined by using two types of fluorescent probes: (a) beta-parinaric acid, a naturally occurring molecule, and (b) 8-anilino-1-naphthalene sulfonic acid, a synthetic organic fluorophore. A computer-centered spectrofluorimeter capable of simultaneous measurement of absorbance, absorbance-corrected fluorescence, and relative fluorescence efficiency was utilized for on-line measurement of all fluorescence parameters. Plots of absorbance corrected fluorescence or of relative fluorescence efficiency versus temperature revealed the same five characteristic temperatures with both types of probe. These characteristic temperatures were independent of the phospholipid composition of the LM suspension cell membranes or their extracted lipids. Plasma membranes, microsomes, and mitochondria containing analogue phospholipids had similar (+/- 1 degree) characteristic temperatures. The presence of analogue phopholipids altered the binding characteristics of beta-parinaric acid with plasma membranes and plasma membrane lipids of LM suspension cells. The equilibrium dissociation constant of plasma membranes and plasma membrane lipids was decreased 2- and 5-fold, respectively, when the cells had been supplemented with ethanolamine. The minimum number of phospholipid molecules per probe binding site was approximately constant in the intact plasma membrane but increased (2-fold) in the isolated plasma membrane lipids. The presence of analogue phospholipids also altered the interaction of 8-anilino-1-naphthalene sulfonic acid with LM cell membranes. The equilibrium dissociation constant of this probe interacting with mitochondrial lipid was decreased 40% by ethanolamine supplementation. The fluorescent properties of both probes were sensitive to the degree of methylation of the polar head group. The absolute values of absorbance-corrected fluorescence and relative fluorescence efficiency were different for each type of membrane from LM cells even with the same analogue supplement. Thus, it appears that LM cells maintain the characteristic temperatures which are a measure of the physical properties of their membranes, despite large alterations of the phospholipid polar head group composition.