The catabolic site(s) of plasma lipoproteins and apolipoproteins has not been established. We have purified one of the major apolipoproteins and explored its catabolic rate and its site of catabolism. Apolipoprotein (Apo) A-1 was purified by column chromatography of canine high-density lipoprotein (HDL) subfraction, HDL3, which was totally delipidated; the purified Apo A-1, molecular weight 28,000, had only one precipitin line to either anti-Apo A-1, or anti-Apo HDL3 serum. Apo A-I was then iodinated, its catabolic rate measured, and the various organs and the subcellular sites of liver involved in the catabolism were investigated. The T 1/2 of 125I-Apo A-I in HDL3 was 3.33 +/- 0.08 days and in plasma 3.52 +/- 0.17 days. The disappearance curve of radioactivity recovered in density fractions d less than 1.110 and d greater than 1.210 Gm. per milliliter was the same as that in HDL3, suggesting identical Apo A-I catabolism in each density class. The liver and kidney absorbed more radioactivity than did the spleen, heart, lung, and intestine. In subcellular fractions of liver, radioactivity was predominantly recovered in the light mitochondrial fraction. There was a statistically significant correlation between the acid phosphatase relative specific activity and the relative specific radioactivity in each subcellular fraction examined at several time intervals after the injection of 125I-Apo A-I. Direct organelle isolation and demonstration of labeled apoplipoprotein uptake indicate that liver lysosomes may play an important role in the catabolism of Apo A-I, as may those of the kidney.