1. The relationship between atherosclerosis development and changes in arterial fatty acid binding protein (FABP) activity was investigated in the aortas of New Zealand rabbits which were fed an atherogenic diet containing 1% cholesterol and 3% peanut oil for 16 weeks. 2. At 4-week intervals, FABP activity, cholesterol and microsomal acylCoA:cholesterol acyltransferase (ACAT) activity were determined in aortic tissue and serum cholesterol was measured; age-matched normal rabbits served as control comparators. 3. Serum cholesterol increased from 35 mg/dl in the normal rabbits to 2290 mg/dl in the 16-week cholesterol-fed rabbits. 4. The microsomal fraction isolated from cholesterol-fed rabbit aortas exhibited a progressive elevation in ACAT activity as time on the diet increased. By 12-16 weeks, ACAT activity had increased approximately 10-fold relative to normal activity. 5. Arterial cholesterol content of the cholesterol-fed animals increased from less than 2 mg/g wet weight to greater than 10 mg/g wet weight at 12 and 16 weeks. In contrast, arterial FABP activity gradually decreased with time on the cholesterol diet; a significant decrease (P less than 0.05) was observed at 16 weeks, where palmitoyl CoA binding was decreased from 61.0 to 36.3 pmol/mg protein. 6. In the cholesterol-fed rabbits, total arterial cholesterol and ACAT activity showed a significant (P less than 0.05) inverse correlation to FABP activity with correlation coefficients of -0.93 and -0.95, respectively. 7. Additionally, FABP activity increased significantly (P less than 0.05) in the 16-week normal rabbit as compared to the 4-week normal rabbit, suggesting an age-dependent interaction.