Goals of the present study were to compare the hemolytic activities of mouse C4 and Slp in a homologous system and to study a possible interaction between these proteins during complement activation. As reagents for mouse C4 and Slp, we used serum of C4(- / -) knockout C57BL / 6 (C4(-) / Slp(-)) mice and sensitized rabbit erythrocytes as target cells. Sera to be tested contained none, either of the two or both proteins. We found that C4(-) / Slp(+) serum has some hemolytic C4 activity, but less than C4(+) / Slp(-) serum. Comparing C4 activities of C4(+) / Slp(-) and C4(+) / Slp(+) sera, we found a threefold enhanced activity in double-positive serum. Hemolytic C4 levels of mixtures of solely C4- and Slp-sufficient sera did not overlap with expected C4 levels, but rather these sera showed synergy. This explains the enhanced activity of double-positive serum. Similar results were observed for total complement activation. In conclusion, Slp has measurable, but poor C4 activity as compared with mouse C4. Using our homologous system, we showed that the enhanced classical pathway activity of double-positive sera is most probably based on synergy between C4 and Slp. Our results answer an old question as to why C4(+) / Slp(+) mice have higher complement levels than C4(+) / Slp(-) mice.