The influence of the initial substrate to microorganisms ratio (So/Xo) on the inhibition of the methane production caused by 4-chlorophenol, 4CP, was studied. The effect of the 4CP on glucose degradation was evaluated measuring the quantity of methane produced during the test. One part of the inoculum was used as it came from its origin and another was fed with ethanol in order to maintain its activity. The inhibition tests were carried out using the same initial concentration of glucose, three initial suspended solids concentrations and eight initial concentrations of 4CP. The use of ethanol-acclimatized sludge presented a great influence on the inhibition results, since the inhibition was greater in the raw sludge than the acclimated one. Significant differences in the inhibition tests were found depending on the initial So/Xo ratio with respect to the glucose as substrate. It was observed that the inhibition decreases as the So/Xo decreases, in other words as the initial biomass concentration increases. When the results were analyzed in respect to the 4CP it was observed that the inhibition increases as the So/Xo increases. Also, it was observed that for the same value of So/Xo there is an increase of the inhibition when the quantity of Xo decreases.