This work aimed to evaluate the effect of different alternative substrates in the production of lettuce and cabbage seedlings in an organic system. The work was conducted in the experimental area of Universidade Estadual de Mato Grosso do Sul, city of Gloria de Dourados, Mato Grosso do Sul state, in plastic greenhouse. The experimental outline used was entirely randomized, with four repetitions. The evaluated treatments were: T1: commercial substrate (Bioplant®), T2: compost of cattle manure, T3: vermiculite, T4: coffee straw, T5: compost of cattle manure (50%)+vermiculite (50%), T6: compost of cattle manure (75%)+vermiculite (25%), T7: compost of cattle manure (25%)+vermiculite (75%), T8: compost of cattle manure (50%)+coffee straw (50%), T9: compost of cattle manure (75%)+coffee straw (25%), T10: compost of cattle manure (25%)+coffee straw (75%), T11: vermiculite (50%)+coffee straw (50%), T12: vermiculite (75%)+coffee straw (25%), T13: vermiculite (25%)+coffee straw (75%), T14: compost of cattle manure (50%)+vermiculite (25%)+coffee straw (25%), T16: compost of cattle manure (25%)+vermiculite (25%)+coffee straw (50%). By the results got, the utilization of substrate made with cattle manure mixed in proportions with the vermiculite introduces itself as an alternative in commercial substrate in production of lettuce and cabbage seedlings in trays system.