The molecular basis for variation in the Mr of the C4 alpha-chain of the fourth component of murine complement from several strains was investigated. All strains were capable of incorporating radiolabeled mannose into the alpha-chain of C4 from peritoneal macrophage cultures. In most cases, carbohydrate was found on both alpha-chain autolytic fragments produced by denaturation of the native C4. However, mice bearing the H-2 haplotypes w7, w16, and w19 produce C4 with no carbohydrate on the larger (COOH-terminal) autolytic fragment. This lack of carbohydrate is sufficient to cause the difference in Mr seen in the C4 alpha-chain from these mice. The chemical removal of all carbohydrate abrogated this difference. The levels of C4 antigen in the plasma of mice from these three strains are normal while the levels of hemolytic activity are reduced 60-80%. This suggests a strong role for carbohydrate in the functional properties of C4.