It was previously reported that free iron was released from rat red blood cells in both an in vitro study with a metabolite, mono-n-butylphthalate (MBP), of di-n-butylphthalate (DBP) and an in vivo study with oral DBP, inducing rat testicular atrophy in hypoxic conditions due to MBP-induced hemoglobin deprivation. This study investigated whether mono-n-alkylphthalates (alkyl-carbon C₄-C₆ ; MAP), which result in rat testicular damage, induce iron release from hemoglobin by incubation with rat red blood cells. Iron release was observed with C₃-C₆ MAP incubation at lower doses and with C₁ and C₂ MAP incubation at higher doses, but not with C₇, C₈, and C₄₍₆₎ MAP.