Anaerobic fungi were isolated from both the rumen and faeces of nine sheep and a cow. A reliable and simple method for the isolation of anaerobic fungi using 24 h rumen incubated milled straw as the inoculum source was developed. We also evaluate the use of chitin measurements as an assay of rumen fungal biomass. Chitin levels were determined from various sample sources (milled barley straw used as the fungal culture substrate in vitro; plant particulate digests from the rumen (PLP) and centrifuged strained rumen fluid (CSRF) using both HPLC and colorimetric methods. Both methods were highly correlated and consequently the simpler colorimetric method was adopted for subsequent studies. There was also a high degree of correlation between anaerobic fungal cellulase activities with the assayed chitin content of milled barley straw cultures over 12 d of an in vitro experiment. The colorimetric chitin assay protocol was then used to assess the diurnal variation and abundance of rumen fungi in in vivo assays. We assessed the distribution of chitin (mg g−1 dry matter) in various fractions of the strained rumen fluid (SRF) and PLP samples from the rumen of sheep. Chitin was detected in all fractions of strained rumen fluid but the main source of chitin in the samples may be attributed to the fungal biomass. We did not detect any significant differences in chitin levels over a 24 h sampling period. Finally, an SEM study on subsamples of milled straw and plant particulate matter used in the chitin assays, revealed that the pattern of the fungal development on substrate material differs from the culture medium to the rumen.