Although methyl iso- and anteiso-branched fatty acids occur widely in the membrane lipids of prokaryotic microorganisms, relatively little is known about the physical properties of phospholipids containing these fatty acids. We report here a monolayer and differential scanning calorimetric characterization of several synthetic phosphatidylcholines containing branched-chain fatty acids, and describe the interactions of these phospholipids with cholesterol and with a bacterial hopanoid. We find that monolayers as well as bilayers of methyl isobranched- and especially of methyl anteisobranched-fatty-acid-containing phosphatidylcholines exhibit a reduced solid-to-fluid phase transition temperature in comparison with linear saturated fatty acid-containing phosphatidylcholines of comparable chain length. We also find that the liquid-condensed or gel states of branched-chain fatty acid-containing phosphatidylcholines are partially disordered relative to those of phospholipids containing linear saturated fatty acids, although the presence of a methyl branch has only a small effect on hydrocarbon chain packing in the liquid-expanded or liquid-crystalline states. The presence of cholesterol was found to produce a marked condensation of liquid-expanded films and a small condensation of liquid-condensed films, whether the phosphatidylcholine contained linear or branched-chain fatty acyl constituents. The presence of a bacterial hopanoid produced similar, although slightly smaller, monolayer-condensing effects, indicating that these compounds may perform a cholesterol-like function in bacterial membranes.