Apoprotein C-II (ApoC-II) functions as a modulator of the hydrolysis of lipoprotein triglycerides by increasing the activity of lipoprotein lipase. Apoprotein C-III (ApoC-III) appears to have an inhibitory effect on ApoC-II-stimulated hydrolysis. Because of the physiologic importance of these apoproteins, we have developed double antibody radioimmunoassays that are capable of measuring the levels of ApoC-II and ApoC-III in human whole plasma and in isolated lipoproteins. ApoC-II and ApoC-III were isolated from very low density lipoproteins by column chromatography. Fractions that were homogeneous on polyacrylamide disc gel electrophoresis and isoelectric focusing and that had the appropriate amino acid compositions were used as antigens, assay standards, and labels. The assays contained ¹²⁵I-ApoC-II or ¹²⁵I-ApoC-III (Na¹²⁵I + lactoperoxidase), rabbit antihuman ApoC-II (or ApoC-III), samples of plasma, lipoproteins, or apoprotein standards, goat antirabbit IgG, and 0.05 M barbital pH 8.6, 0.01% Triton X-100. The assays had the specificities, accuracies, precisions, and immunologic indentities between standards and samples required of all such assays. All of the ApoC-II in plasma in isolated lipoproteins was detected by the ApoC-II assay. ApoC-III₁ and ApoC-III₂ were equally reactive, and all of the ApoC-III₀, ApoC-III₁, and ApoC-III₂ in plasma and in lipoproteins was detected by the ApoC-III assay. ApoC-II and ApoC-III levels were measured in 171 fasting plasmas taken from normal subjects and patients with type II–V hyperlipidemia. “Normal” values for white subjects were approximately 4 and 16 mg/dl for ApoC-II and ApoC-III, respectively. Levels were several fold elevated in the hyperlipidemias. In normal subjects ∼40% of ApoC-II and ApoC-III were found in the d < 1.006 fraction, ∼15% in d = 1.006–1.063, ∼45% in the d = 1.063–1.21. These distributions were altered in hyperlipidemia. However, in all cases the d>1.21 fraction contained < 5% of either ApoC-II or ApoC-III.