The concentration of nitrate-nitrogen in cotton petioles was directly related to the rate of nitrogen applied and was greatest at the earliest sampling date, decreasing as the season progressed. There was an increase in the degree of association between the level of nitrogen applied and petiole nitrate-nitrogen, and between petiole nitrate-nitrogen and final yield, up to the time of early flowering, when peak correlation coefficients were reached, after which there was a decline. The level of petiole nitrate-nitrogen showed high positive correlations with the rate of nitrogen application as well as with yield. The final yield showed a significant, positive and linear regression on the level of petiole nitrate at the time of early flowering. A close relationship was also shown to exist between a critical concentration, represented by a narrow range of nitrogen levels in the neighbourhood of 2000 parts per million, and final cotton yield. Yield reductions were related to the number of days petiole nitrogen remained below the critical concentration. The use of petiole analysis as a guide in the nitrogen nutrition of cotton, in the evaluation of various experimental treatments and as an aid in interpreting research results, was demonstrated.