A quantitative profile of cytochalasin D production by Xylaria arbuscula was followed by growing the fungus in rice, Czapek, Czapek enriched with yeast extract, wheat, and corn. This cytochalasin producer, X. arbuscula, was collected as an endophytic fungus from healthy tissues of Cupressus lusitanica (Cupressaceae). A new HPLC method was developed using a synthetic N-acetyl-L-phenylalanine ethyl ester as internal standard, which showed a good correlation coefficient (r2 = 0.9995). The results varied from 6.40 to 39.55 mg per 100 g of culture medium, with wheat being the best medium for cytochalasin D production. The level of any free amino acids in the medium, not necessarily phenylalanine, appeared to be an important factor to enhance cytochalasin D biosynthesis.