Four rumen-fistulated cows were fed concentrate:corn silage (40: 60) diets in a 4 × 4 latin square design experiment. The diets differed in the soybean meal or soybean meal treated with 0.1, 0.3 or 0.6% (wt/wt) formaldehyde. Rumen contents were collected hourly from 0900 h to 1600 h with an additional sample at half an hour after the 1100 h feeding on 3 days in the second week of each 2-wk experimental period. The maximum ammonia concentration in rumen contents decreased with increasing levels of formaldehyde used to treat the soybean meal. From 1130 to 1200 h the rates of ammonia appearance in the rumen contents were different (P < 0.05) for all four diets. Treatment of soybean meal with between 0.1 and 0.3% (wt/wt) formaldehyde reduced its in vitro degradation by rumen protease by about 50% compared with the untreated control. The rates of in vivo ammonia appearance and in vitro proteolysis were highly correlated (r = 0.966, P < 0.01) between 1130 h and 1200 h. These rates of rumen ammonia appearance reflected the in vivo rate of proteolysis. Both bacterial and protozoal protein content decreased significantly at the higher levels of formaldehyde treatment. There was a negative correlation (r = − 0.63, P < 0.01) between protozoal and bacterial protein. Key words: Formaldehyde, soybean meal, bovine, rumen, degradation, protease