The elicitation of 24-h contact sensitivity (CS) in mice requires a serotonin-dependent, 2-h response called CS initiation. We studied the role of complement (C) by comparing CS in DBA/1 (C5-normal) vs DBA/2 (C5-deficient) mice and found impaired 2-h, but not 24-h, CS. We showed previously that 2-h responses represent CS initiation and are required for elicitation of 24-h responses. Treatment of C5-deficient mice with absent macroscopic responses (ear swelling) with a selective serotonin antagonist inhibited 24-h CS, suggesting that C5-deficient mice had submacroscopic (i.e., microscopic), serotonin-dependent CS initiation. When normal mouse serum was used as a source of C5 to reconstitute C5-deficient mice, significant 2-h responses were restored. Furthermore, heat treatment of normal mouse serum to inactivate C abrogated restoration of 2-h responses. Thus, C5 was suggested to be involved in CS initiation. Using a suboptimal immunizing dose of Ag revealed an impaired 24-h component of CS in DBA/2 mice, but not in DBA/1 mice, and also in C5-deficient B10.D2/o mice compared with C5-normal B10.D2/n mice with a suboptimal eliciting dose of Ag. Again, reconstitution of B10.D2/o mice with normal mouse serum restored deficient 24-h CS responses. Thus, 2-h and classical 24-h CS probably depend in part on C5. These results imply that C5 may play a role in the elicitation of 24-h CS, probably via required preceding CS initiation.