Fungal leaf endophytes are a potentially large reservoir of diversity that has yet to be fully explored. Recent observations indicate that cottonwoods (Populus spp.) appear to be an exception to the apparent ubiquity of these fungi. This observation could result from unculturable endophytes, lack of viable inoculum, or the action of antifungal phytochemicals. To investigate these hypotheses I conducted a study of the fungal endophytes of Populus fremontii in a riparian, gallery forest in central Arizona. No fungi grew from cultured asymptomatic leaf segments. Ruling out the possibility of unculturable endophytes, I observed no fungal structures in microscopic examinations of cleared and stained leaf segments. To examine the presence of viable fungal inoculum, I cultured inoculum from the air column beneath mature canopies, the surface of asymptomatic leaves and 2^nd year stems. Fungal colony forming units (CFU) grew in 100% of aerial and leaf surface inoculum cultures and 40% of 2^nd year stem cultures. When added to the agar medium, aqueous leaf extract significantly reduced the number of CFU by 89.5% in aerial and 76.3% in surface inoculum cultures. These results indicate that fungal endophytes are unusually absent in cottonwood leaves, possibly due in part to antifungal phytochemicals. Historically large negative impacts of fungi, especially rusts, may have selected for the development of highly effective barriers to fungal colonization and led to the wholesale rejection of cottonwood fungal leaf endophytes. Investigating cottonwood phytochemicals that inhibit fungi may lead to the discovery of useful antifungal compounds and a better understanding of the evolutionary ecology of cottonwood-fungus interactions.