A better understanding of the effect of soil nitrogen (N) level on cotton plant carbon metabolism is important in cotton research and production. Field studies were conducted at two locations of Clarkedale (Northeast Arkansas) and Fayetteville (Northwest Arkansas) in 2000 to investigate cotton plant physiological and yield responses to two levels of soil N fertilizer applications. The experiments consisted of two treatments of high N (HN) and low N (LN). The HN and LN treatments received a total of 100 and 50 (Clarkedale) or 100 and 40 (Fayetteville) lb. N/A, respectively. At Clarkedale, LN decreased plant height, leaf area index, plant dry matter accumulation, leaf chlorophyll concentration and leaf net photosynthetic rate during fruiting. As a result, the LN treatment had significantly lower boll numbers and lint yield than the HN treatment. Leaf nonstructural carbohydrate concentrations changed tremendously with growing season, but did not differ between the two N treatments at most measuring times. At Fayetteville, there were no statistical differences between HN and LN treatments in plant growth, lint yield and most physiological measurements. The inconsistent results in the two locations might be associated with soil fertility levels because the soil N fertility before planting at Fayetteville was much higher than at Clarkedale.