Pyrroloquinazolinediamine (PQD) derivatives such as tetra-acetamide PQD (PQD-A4) and bis-ethylcarbamyl PQD (PQD-BE) were much safer (with therapeutic indices of 80 and 32, respectively) than their parent compound, PQD (therapeutic index, 10). Further evaluation of PQD-A4 and PQD-BE in single and multiple pharmacokinetic (PK) studies as well as corresponding toxicity studies was conducted with rats. PQD-A4 could be converted to two intermediate metabolites (monoacetamide PQD and bisacetamide PQD) first and then to the final metabolite, PQD, while PQD-BE was directly hydrolyzed to PQD without precursor and intermediate metabolites. Maximum tolerant doses showed that PQD-A4 and PQD-BE have only 1/12 and 1/6, respectively, of the toxicity of PQD after a single oral dose. Compared to the area under the concentration-time curve for PQD alone (2,965 ng.h/ml), values measured in animals treated with PQD-A4 and PQD-BE were one-third (1,047 ng.h/ml) and one-half (1,381 ng.h/ml) as high, respectively, after an equimolar dosage, suggesting that PQD was the only agent to induce the toxicity. Similar results were also shown in multiple treatments; PQD-A4 and PQD-BE generated two-fifths and three-fifths, respectively, of PQD concentrations, with 8.8-fold and 3.8-fold safety margins, respectively, over the parent drug. PK data indicated that the bioavailability of oral PQD-A4 was greatly limited at high dose levels, that PQD-A4 was slowly converted to PQD via a sequential three-step process of conversion, and that PQD-A4 was significantly less toxic than the one-step hydrolysis drug, PQD-BE. It was concluded that the slow and smaller release of PQD was the main reason for the reduction in toxicity and that the active intermediate metabolites can still maintain antimalarial potency. Therefore, the candidate with multiple-step hydrolysis of PQD could be developed as a safer potential agent for malaria treatment.