Ruminal microorganisms, preserved either lyophilized or frozen, were compared with freshly strained ruminal fluid for proteolytic activity and as inoculum source for determination of ruminal protein degradation rates by the inhibitor in vitro method. Dialysis and glycerol addition had no effect on the proteolytic activity of preserved microorganisms. Net release of NH3 and total amino acids from protein using the fluid plus particle-associated microorganisms was higher than that found using the fluid-associated microorganisms alone. Method of inoculum preservation altered total proteolytic activity, but harvesting bacteria using centrifugal force greater than 5,000 x g did not increase proteolytic activity of the pellet. The proposed method for harvesting and preserving microorganisms consisted of centrifuging strained ruminal fluid at 5,000 x g (30 min at 4 degrees C), stirring the pellet in a 50:50 (vol/vol) solution of glycerol-McDougall's buffer for 15 min, and then storing at -20 degrees C. Protein degradation rates in incubations with preserved microorganisms were four to eight times slower than when using fresh ruminal fluid; however, feed proteins were ranked similarly for degradation rate. Preincubating the preserved microorganisms reduced blank concentrations of NH3 and total amino acid and increased protein degradative activity of the preserved inoculum. Degradation rates with preincubated, preserved inocula were similar to those obtained using fresh ruminal fluid. These results indicated that mixed ruminal microorganisms can be preserved by freezing and, after a preincubation period of 6 h, used as the inoculum source for in vitro estimation of ruminal protein degradation.