Acremonium coenophialum, a fungal endosymbiont in tall fescue, is responsible for the production of alkaloid toxins. Animals grazing endophyte-infected tall fescue often show toxicosis. In marginal environments, the endophyte is important for long-term survival of tall fescue. Few differences in endophyte isolates from individual tall fescue plants have been reported. To aid development of a toxicosis-free tall fescue, it is important to identify differences in endophyte isolates. This report describes variation in nitrogen utilization in a defined culture medium by endophyte isolates from Kentucky-31 tall fescue. Overall, the best nitrogen sources for dry weight production of mycelium were proline and potassium nitrate. Thirty-four isolates grown on agar-solidified defined media with single nitrogen sources showed variation in nitrogen utilization. Fifty percent of the isolates were unable to utilize two or more amino acids. Manipulation of endophyte variation could lead to development of a toxicosis-free tall fescue cultivar.