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a b s t r a c tThe effects of dietary substitution of soybean meal (SBM) with ethanol-treated jatropha kernel cake with seed shells (TJC) on nutrient intake and digestibility, nitrogen balance, pH, rumen ammonia nitrogen (NH 3 -N), serum levels of alanine aminotransferase (ALT, EC 2.6.1.2) and aspartate aminotransferase (AST, EC 2.6.1.1), and ruminal degradation kinetics of SBM and TJC crude protein (CP) in Holstein heifers were evaluated. Four rumen-cannulated heifers were distributed in a 4 Â 4 Latin square design, [body weight (BW) ¼ 379 72.13 kg]. Each experimental period lasted 15 days, with eight days for diet adaptation and seven days for data collection. The heifers were fed four complete isonitrogenous diets containing 65% corn silage and 35% concentrate on a dry matter (DM) basis. The treatments consisted of four levels of SBM substitution for TJC þurea/ammonium sulfate: 0, 33, 67, and 100%, on a DM basis. Ethanol treatment of the jatropha kernel cake removed 4 99% of the phorbol esters. The effective degradability of the CP for TJC was 425.6 g/kg. Intakes of DM, organic matter (OM), CP, ether extract (EE), neutral detergent fiber (NDF) and total digestible nutrients (TDN) decreased linearly with the substitution of SBM with TJC. The DM intakes were 18.0, 16.2, 16.8 and 11.3 g/kg BW for 0, 33, 67 and 100% substitution, respectively. There was no effect of the TJC inclusion level on nutrient digestibility. The rumen pH increased linearly with increasing substitution levels. The rumen NH 3 -N was not affected by the inclusion of jatropha (7.79 mg/dL overall mean). The amounts of ingested and absorbed nitrogen (N) and the N-balance decreased linearly with increasing TJC levels. The serum levels of ALT and AST were not affected by the inclusion of TJC in the diets. Despite the decrease in phorbol esters, ethanol treatment of jatropha kernel cake was not enough to promote adequate nutrient intake by the heifers.