The essential fatty acids, particularly arachidonic, are important components of intracellular membrane systems. Their absence during dietary manipulation has been postulated to cause alterations in both composition and function of membranes and associated enzyme systems. In the current investigation, the effect of essential fatty acid deficiency on the induction of hepatic microsomal membranes my phenobarbital was studied. Control rats were fed a standard chow diet and either injected daily for 4 days with phenobarbital or with a placebo. Experimental animals were given an essential fatty acid-deficient (EFAD) diet and similarly injected with either phenobarbital or placebo. Following the above regimens, liver tissue was obtained for electron microscopy and biochemical membrane analysis. Control animals given phenobarbital displayed a marked proliferation of smooth endoplasmic reticulum in comparison to placebo controls. In contrast, EFAD rats did not exhibit an endoplasmic reticulum response to phenobarbital injection and appeared to recover from the drug administration injection more slowly than control animals. The alterations in fatty acid composition characteristic of an EFAD diet were observed in the microsomal membranes of the deficient animals. The concentrations of palmitic, palmitoleic, oleic, and 5,8,11-eicosatrienoic acids were significantly increased above that found in control animals. Concentrations of linoleic and arachidonic acids were reduced. Phenobarbital administration significantly increased the concentrations of palmitic, linoleic, and arachidonic acids in microsomal fractions of chow-fed animals but not in EFAD animals. Conversely, eicosatrienoic acid concentrations increased in phenobarbital-treated EFAD animals but not in chow-fed, drug-injected rats. The triene/tetraene ratio reflected these alterations. Essential fatty acid deficiency causes biochemical alterations of the hepatic microsomal membranes which are exaggerated by the administration of phenobarbital. These compositional changes appear to alter the functional ability of the membranes to proliferate in response to phenobarbital and, thereby, inhibit the efficaceous metabolism of this drug.