Using regiospecifically oxygen-18-labeled antimalarial 1,2,4-trioxane 1, previous studies showed ferrous ion reduces the crucial peroxide linkage to form oxy radical and then carbon radical intermediates leading to the C4-hydroxylated product 2 and the ring-contracted product 3, with only the pathway involving a C4 radical intermediate leading to 2 important for high antimalarial activity. Now, significant further evidence supporting the key role and limitations of such C4 radicals in the antimalarial activity of several new tricyclic trioxanes bearing diverse substituents at C4 is reported. The antimalarial data in Table 1 support generalizations: (1) C4β-substituted derivatives (e.g., 5, 6, 7) with stereochemistry allowing critical H atom transfer to form C4 radicals are 12-200 times more active than corresponding α-substituted derivatives; (2) tertiary C4 radicals (from β-substituents) in derivatives like 5 and 6 are more potent than secondary C4 radicals, being comparable to artemisinin and 11-13 times more active than C4-unsubstituted parent 4; (3) C8-unsubstituted C4-benzyl analog 9 has significantly higher activity than parent analog 8; (4) unexpectedly, C4β-substituents that overly stabilize adjacent carbon radicals (e.g., 7, 10) reduce antimalarial activity. Study of 10's reaction with ferrous ions showed it forms only ring-contracted acetal 11 (no C4-hydroxylated product), unlike active analog 5 (1:4 ratio of 2-like and 3-like products), suggesting overly stable radicals shunt to the lower pathway avoiding C4 radical formation. In conclusion, these results further support the importance of a C4 carbon-centered radical leading to a C4 hydroxylated product for high antimalarial activity of 1,2,4-trioxanes, while showing that increasing the stability of such a radical beyond that of a tertiary radical reduces potency. These structure-activity relationships and mechanistic understanding may help design better antimalarial trioxanes.