Aims: To investigate, using culture-independent methods, whether the ruminal bacterial structure, population and fermentation parameters differed between sampling locations and time. Methods and Results: The detectable bacteria and fermentation parameters in the digesta from five locations in the rumen of three cows at three time points were analysed. The PCR-denaturing gradient gel electrophoresis (PCR-DGGE) profiles were similar among digesta samples from five locations (95 center dot 4%) and three time points (93 center dot 4%) within cows; however, a lower similarity was observed for samples collected from different host animals (85 center dot 5%). Rumen pH and concentration of volatile fatty acids (VFA) were affected by time points of sampling relative to feeding. Conclusions: The detectable bacterial structure in the rumen is highly conserved among different locations and over time, while the quantity of individual bacterial species may change diurnally in response to the feeding. Significance and impact of the study: This study supplies the fundamental understanding of the microbial ecology in the rumen, which is essential for manipulation of ruminal microflora and subsequent improvement in animal production.