Chimeric proteins using membrane cofactor (CD46) and decay accelerating factor (DAF or CD55) were generated to further investigate the functional domains involved in the regulation of human serum complement. Following activation of the classical pathway, the isolated substitution of CD46 SCR III (x3DAF) exhibited a modest regulatory activity comparable to that of CD46. The isolated substitution of CD46 SCR IV (x4DAF), and the combined CD46 SCR III+IV substitutions (x3/4DAF) were essentially as efficient as DAF. No regulation of C3b deposition was observed with the combined CD46 SCR I+II substitutions (x1/2DAF). When tested after activation of the alternative pathway, both the x3DAF and x3/4DAF chimeras failed to regulate C3b deposition, while the x4DAF chimera still displayed some activity. In contrast to that observed following classical pathway activation, the x1/2DAF chimera exhibited a similar efficiency to wild type CD46 and DAF in controlling C3b deposition. Using SCR specific antibodies, the regulatory activity of the x1/2DAF chimera against the alternative pathway was mapped to the first three distal SCR (i.e. DAF 1, DAF 2 and CD46 III). These data demonstrate that several combinations of SCR domains from two related complement regulators can result in functional molecules, and reveal a novel and cryptic functional role for DAF SCR1.