A Palmer amaranth (Amaranthus palmeri S. Watson) population (KCTR: KS Conservation Tillage Resistant) collected from a conservation tillage field was confirmed with resistance to herbicides from at least six sites of action, including 2,4-D. The objectives of this research were using KCTR A. palmeri to investigate i) the level of 2,4-D resistance, ii) 2,4-D absorption and translocation profiles, iii) the rate of 2,4-D metabolism compared with 2,4-D-tolerant wheat (Triticum aestivum L.), and iv) the possible role of cytochrome P450s (P450s) in meditating resistance. Dose-response experiments were conducted to assess the level of 2,4-D resistance in KCTR compared to susceptible plants, KSS (KS 2,4-D susceptible) and MSS (MS 2,4-D susceptible). KSS, MSS, and KCTR plants were treated with [14C] 2,4-D to determine absorption, translocation, and metabolic patterns. Additionally, whole-plant dose-response assays were conducted by treating KCTR and KSS plants with P450-inhibitors (malathion, piperonyl butoxide: PBO) prior to 2,4-D application. Dose-response experiments indicated a 6 to 11-fold 2,4-D resistance in KCTR compared to susceptible plants. No difference was found in percent [14C] 2,4-D absorption among the populations. However, 10% less and 3 times slower translocation of [14C] 2,4-D was found in KCTR compared to susceptible plants. Importantly, [14C] 2,4-D was metabolized faster in KCTR than susceptible plants. At 24, 48, and 72 hours after treatment (HAT), KCTR metabolized ∼20-30% more [14C] 2,4-D than susceptible plants. KCTR plants and wheat generated metabolites with similar polarity. Nonetheless, at 24 HAT, ∼70% of [14C] 2,4-D was metabolized in wheat, compared to only 30% in KCTR A. palmeri. Application of malathion prior to 2,4-D increased the sensitivity to 2,4-D in KCTR, suggesting involvement of P450s in mediating 2,4-D metabolism. However, no such impact of PBO was documented. Overall, this study confirms that enhanced metabolism is the primary mechanism of 2,4-D resistance in KCTR.