AbstractA series of experiments was undertaken to determine population statistics forin vitroorganic matter digestibility (in vitroOMD) data and to examine the effects of basal diet, donor animal and precollection fasting interval on the activity and specificity of rumen fluid inoculum. The experiments utilized wether sheep, a diverse set of pasture grass and legume feeds prominent in the Australian subtropics and the Tilley and Terryin vitrodigestibility procedure running under the operating pressure of a practicing feeds evaluation laboratory.The standard errors ofin vitroOMD estimates for within and between batch runs were ±0·88 × 10−2and 0·62 × 10−2, respectively. These error terms were used to develop protocols to accept, reject or scale rawin vitroOMD data. Differences between donor animals in the activity of rumen fluid were highly significant. Extending the precollection fasting interval beyond 16 h was associated with a substantial decline in inoculum activity.Anin vitro‐in vivocalibration relationship based on fifteen test feeds and using lucerne (Medicago sativa) as basal diet was described by the linear model y = 1·3 x‐0·195±4·9 × 10−2r= 0·79 (y =in vivoOMD, x =in vitroOMD). Despite large effects of basal diet on both the absolute values and relative ranking of test feeds, neither the RSD norrvalues were improved using alternative diets to Lucerne chaff.The results highlight the need to formally standardize the analytical and biological components of thein vitrodigestibility procedure to safeguard t