Flavonoids have been studied extensively because they offer great potential health benefits. In this study, enzymatic hydrolysis of glycosylated quercetin, kaempferol, and naringin was used to obtain their sugar-free aglycones. The investigation also employed a validated HPLC method to obtain the chiral disposition of the aglycone naringenin enantiomers. These analyses were conducted on exocarp, mesocarp, and seed cavity tissues of field-grown tomato (Lycopersicon esculentum Mill.) mutants (anthocyanin absent, atroviolacea, and high pigment-1) and their nearly isogenic parent (cv. Ailsa Craig) at immature green, "breaker", and red ripe maturity stages. Concentrations of all flavonoids using enzymatic hydrolysis were significantly higher than previously reported concentrations using acid hydrolysis. Presumably, this occurred due to a more specific and rapid hydrolysis of the glycoside moiety by the beta-glucosidase enzyme. The glycoside S-naringin was the predominant enantiomer in all fruit tissues, although the aglycones free R- and S-naringenin were detected in both exocarp and mesocarp. Whereas there was significantly more quercetin than kaempferol in exocarp tissue, they were present in about equal concentrations in the mesocarp. Quercetin concentrations were higher in the exocarp and mesocarp of immature green and breaker fruit of the high pigment-1 mutant than in the other genotypes, supporting the observed photoprotection and potential health benefits of the high pigment-1 tomato genotype.