Two hundred fifty-two Hy-Line W36 laying hens were allotted in a randomized block design with 6 treatments, 7 replicates and 6 hens per experimental unit in order to determine the ideal digestible isoleucine: digestible lysine (Ile:Lys) ratio for laying hens aged 24-40 weeks. Experimental diets contained 5.92, 6.33, 6.73, 7.14, 7.54 and 7.95 g/kg of digestible isoleucine, resulting in different Ile:Lys ratios (0.73:1, 0.78:1, 0.83:1, 0.88:1, 0.93:1 and 0.98:1). Experimental diets were isonitrogenous, with different dietary contents of glutamic acid, and made isocaloric by adjusting the levels of cornstarch. All essential amino acids were provided proportionally to digestible lysine. Feed intake, egg production, egg weight, egg mass, feed conversion ratio per egg mass, feed conversion ratio per dozen eggs, albumen, yolk and eggshell content were assessed. Feed intake, egg production, egg mass and feed conversion ratio were influenced by Ile: Lys ratios quadratically. The egg weight was not affected by Ile: Lys ratio. No differences were observed in the egg quality according to Ile: Lys ratios. The data indicates that the optimum digestible isoleucine: digestible lysine ratio for laying hens aged 24-40 week is 0.84:1, corresponding to the consumption of 681 mg of isoleucine and 811 mg of lysine/hen day-1.