Chronic ethanol feeding has been shown to decrease the number of functionally active mitochondrial ribosomes by 55%. In this work, 55S mitochondrial ribosomes were isolated from rat liver and their constitutive proteins characterized by two-dimensional polyacrylamide gel electrophoresis and quantified by densitometry. A total of 86 proteins were found to be associated with the mitochondrial ribosome. This compares with 70 isolated from cytoplasmic ribosomes. In addition, mitochondrial ribosomal proteins were found to be significantly less basic than their cytoplasmic counterparts. Chronic ethanol feeding was found to significantly decrease the levels of a number of constitutive proteins of the mitochondrial ribosome when compared to those isolated from pair-fed controls. Sucrose density gradient analyses revealed a significant decrease in the number of intact 55S ribosomes. It is suggested that ethanol-elicited alterations in specific constitutive proteins of the mitochondrial ribosome may lead to impaired assembly of the monosome and that this may result in lower levels of those displaying functional activity.