The previously described one-step hemolytic assay for the fourth component of complement (C4) that employs C4-deficient (C4D) guinea pig serum was modified to allow assessment of mouse C4. Of a number of variables evaluated including the class and quantity of sensitizing antibody, concentration of C4D serum and metals, ionic strength of the buffer, and the duration and temperature of incubation, substitution of IgG anti-sheep red blood cell antibodies for the standard IgM hemolysin and the use of C4D serum at relatively high concentrations (1 : 20) were necessary to obtain an accurate, reproducible and sensitive hemolytic assay. The sensitivity of the assay could be increased approximately 2-fold by the addition of human C2 and approximately 30% by employing 51Cr-labeled sheep red blood cells. The former probably helped to overcome a partial incompatibility between mouse C4 and guinea pig C2 and the latter permitted the use of 10-fold fewer erythrocytes in the assay system. Employing this assay, C4 activity was determined in H2-congenics and recombinants and the effect of age, sex, and methods of procuring and preserving mouse serum and plasma evaluated. In most strains, males had a 10-30% higher level of C4 hemolytic activity than females, and young (<6 weeks) and older (>20 weeks) mice had lower activity compared to mice between 6 and 12 weeks. Procuring samples in 5 mM EDTA (final concentration) and subsequent storage at -70°C permitted long-term preservation without loss of hemolytic activity. Allowing mouse blood to clot at 4, 22 or 37°C, even for brief periods, was associated with complement activation and depletion of C4 functional activity. C4 assays of H2-congenics and infromative recombinants conclusively demonstrated that this hemolytic activity mapped to the S region of the H-2 complex.