The effect of dietary composition on the RNA and ATP concentrations in rumen bacteria was determined. Four dry Simmental cows received two diets (H, hay only; C, hay and concentrate) and samples of rumen fluid were taken at three different times during the day. Rumen fluid was analysed for DM, N, pH, VFA, RNA, ATP and total bacterial count. There were no significant differences in RNA concentration whether it was determined in fresh or frozen samples or after having been precipitated overnight. The effect of time of sampling was almost always significant: for pH, ammonia nitrogen, propionic acid, n-butyric acid and total VFA this was particularly evident at 12:00 h in comparison with 08:00 and 16:00 h (P < 0·05). Bacterial DM, total count and ATP content decreased linearly with increasing time from the morning meal (P < 0·01), while the highest RNA content was observed at 12:00 h and the lowest at 16:00 h (P < 0·01). The bacterial content (DM basis) of RNA and the RNA:N, RNA:total count and ATP:N ratios were significantly higher with diet C, while differences for ATP (μg mg−1 DM), nitrogen (mg per 100 mg DM) and ATP:RNA ratio were not significant. The effect of sampling time was similar for the two diets: the RNA content of bacterial cells increased from 08:00 h to 12:00 h (P < 0·05), while the RNA:N, RNA:total count and ATP:RNA ratios increased from 08:00 to 12:00 h and decreased from 12:00 to 16:00 h. The ATP content had a similar trend to that of RNA, but differences with time were not significant. Although the use of RNA and ATP as bacterial markers has to be considered with some caution, their determination together with rumen parameters, such as pH, VFA and ammonia, could contribute to useful investigations of the anaerobic fermentative process.