The quantification of archaeol, a methanogen membrane lipid, may provide an alternative method to estimate methanogen abundance. The focus of this study was to determine the location of methanogens in the ruminant digestive tract using this biomarker. Archaeol was quantified in samples obtained from four lactating cows with rumen cannulae that grazed on either white clover (WC) or perennial ryegrass (PRG) in a changeover design study with three 3-week periods. Faeces were collected over the final 5 d of each period and total rumen contents (TRC) were obtained on the final 2 days (day 1: 9 am; day 2: 3 pm). Solid-associated microbes (SAM) and liquid-associated microbes (LAM) were also isolated from the TRC. Concentrations of archaeol in the TRC showed a significant diet by time interaction, which may be related to diurnal grazing patterns and different rumen conditions associated with PRG or WC diets. There was significantly more archaeol associated with SAM than LAM, which may reflect difficulties of methanogen proliferation in the liquid phase. Faeces had higher concentrations of archaeol than SAM and LAM which was unexpected, although, losses of methanogens may have occurred during isolation (i.e. attachment to protozoa and very small particles), or the methanogens associated with SAM may have been underestimated. There was no significant relationship between faecal and TRC archaeol concentrations. Finally, there was a significant positive relationship between rumen pH and concentrations of archaeol in SAM and LAM, which may be caused by pH and/or WC diet effects. In conclusion, archaeol is potentially a useful alternative marker for determining the abundance of methanogens in the ruminant digestive tract. This work has also highlighted the difficulties associated with methanogen quantification from microbial isolates, and the need for more representative rumen sampling in future studies.