In incubations in vitro with rumen fluid, the effect of two methane inhibitors, linseed oil hydrolysate (LOH) and chloral hydrate (CH) on the efficiency of microbiol growth was investigated. Total and net microbial growth were determined from 32PO43- and NH3--N incorporation respectively and expressed as g N incorporated per kg organic matter fermented (gN/kgOMf). In a first series on incubations, it was found that LOH had no influence on overall microbial growth efficiency, while with CH, a small but significant decrease of total and net growth efficiency was measured. Further experiments showed that this was not due to accumulation of hydrogen gas in the CH incubations. Microscopic examination showed a toxic effect of LOH on protozoa, but with CH, no such effect was observed. This observation, together with earlier work where a considerable increase in microbial growth efficiency was found in vitro after defaunation of the rumen suggested the following hypothesis: both inhibitors lowered bacterial growth. In the case of LOH, this effect is marked by the defaunating action of LOH, the latter resulting in an increased growth efficiency of the bacterial fraction. This hypothesis was confirmed by incubations with washed cell suspensions (WCS) of mixed rumen bacteria, where growth efficiency was indeed decreased by both inhibitors. The possible mechanism explaining this phenomenon was discussed.