Cotton petioles from irrigated plants grown under varying nitrogen regimes were analyzed for nitrate‐N (NO3‐N). The most recent, fully matured petioles were selected. The concentration of NO3‐N in the petioles was related to applied N rates and the yields of lint obtained. The concentration of NO3‐N for any given N application declined as the season progressed. The concentration of petiole nitrate increased at all sampling dates as the rate of applied N increased. The relationship between applied N and NO3‐N concentrations was most predictable when samples were collected two weeks after the initiation of squaring. The influence of applied N on the concentration of petiole nitrate was also greatest at this stage. The diagnosis of either N deficiency or excess N would be feasible by petiole analysis when the effects of stage of growth could be separated from the effects of soil N.