Since the mid-1980s, a syndrome known as mature watermelon vine decline (MWVD) has had a serious effect on watermelon (Citrullus lanatus Thunb.) crops in Southern Indiana. As efforts to identify a pathogen responsible for MWVD have been unsuccessful, we have examined cultural conditions that might contribute to the syndrome. Field conditions were simulated in greenhouse pot trials to assess the impact of one or more factors on watermelon growth. Alone, low organic matter, soil acidity, black plastic mulch, and liming did not significantly affect root fresh weight; however, when these conditions were combined, root fresh weight was significantly reduced. Alanap-treated watermelons in combination with simulated cultural conditions resulted in further reduction of root fresh weight and had symptoms similar to MWVD. Watermelon plants grown in Alanap-treated, aged soil (from the previous year's experiments) under combined deficient cultural conditions demonstrated increased symptoms of MWVD and susceptibility to the pathogens Rhizoctonia and Pythium spp. Alanap, N-1-naphthylphthalamic acid (NPA), is a preemergent herbicide that functions as an inhibitor of auxin efflux and is widely used by watermelon farmers to impede obnoxious weeds. Metabolism of Alanap in planta involves aryl amidases (aminopeptidases) that also function in defense responses. We hypothesize that negative cultural practices are likely to inhibit defense responses and watermelon resistance to residual Alanap, leading to MWVD. We suggest that MWVD incidence is increased by certain common cultural conditions and that the incidence of MWVD can be reduced by altering these cultural practices.