A study was conducted for biochemical analysis of ? – carotene and iron content in lettuce (Lactuca sativa L) under different water regime in a naturally ventilated polyhouse erected at the experimental farm of the Deptt. of Vegetable Science, Dr Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan during 2009-10 and 2010-11. Twelve treatment combinations comprising of three depths of irrigation water (1.0, 1.5 and 2.0 cm) and four irrigation intervals (4, 6, 8 and10 days) were replicated thrice in a randomized block design (factorial) having plot size of 1.80 m x 1.80 m with a spacing of 45 X 30 cm. The observations were recorded on ten randomly selected plants on ? – carotene content (?g/100g) and iron content (mg/100g). Analysis of variance showed highly significant differences among different treatments for ?-carotene studied but treatment combination comprising of 1.5 cm of irrigation water applied after 8 days was rated as the best combination. Similarly the maximum value (1.28 mg/100g) of iron content was recorded in the treatment having 2.0 cm depth of irrigation water and minimum (1.22 mg/100g) with 1.0 cm of irrigation water.