In the alternative pathway of complement (APC) factor H is the primary control factor involved in discrimination between potential pathogens. The APC deposits C3b on possible Ags, and the interaction with factor H determines whether the initial C3b activates the APC. Factor H is composed of a linear array of 20 homologous short consensus repeats (SCR) domains with many functional sites. Three of these sites are involved in binding C3b and regulating complement activation; others bind to sialic acid and/or heparin and are responsible for host recognition. Using site-directed mutations we have examined the contributions of each of these sites to target discrimination and to functional activities of factor H. Decay acceleration by SCR1–4 of C3/C5 convertases bound to nonactivators was strongly dependent on SCR domains 11–15 and 16–20. Loss of these regions caused a 97% loss of activity, with SCR16–20 being the most critical (> 90% loss). On APC activators the pattern of site usage was different and unique on each. On yeast, deletion of the 10 C-terminal domains (SCR11–20) had no effect on specific activity. On rabbit erythrocytes, this deletion caused loss of 75% of the specific activity. An examination of binding affinity to C3b on the four cell types demonstrated that factor H exhibits a unique pattern of SCR involvement on each cell. The results reveal a complex molecular mechanism of discrimination between microbes and host in this ancient innate defense system and help explain the different rates and intensities of APC activation on different biological particles.