Past studies indicated that the 2nd generation AjiPro-L (A2G, Ajinomoto Co., Inc.) has higher bioavailability than the 1st generation product (A1G) based on in vivo plasma lysine technique (Whitehouse et al., 2014; Tucker et al., 2014). The objective of this study was to compare the other characteristics between A2G and A1G. Three ruminally fistulated lactating cows fed a diet composed of corn silage, grass silage and concentrates were used to determine in situ rumen stability. A1G and A2G were placed in Nylon bags (1 g per bag, 5 × 7 cm, pore size 53 ± 10 μm) and incubated in the rumen for 6, 12 and 24 h, respectively. After washing and drying bags, lysine (Lys) content in the residual products was analyzed. Three dry cows, fistulated both ruminally and duodenally and fed a corn silage based diet, were used to determine rumen-bypassed and fecal excreted Lys from each product. A1G or A2G was ruminally administrated along with the highly protected l-arginine (HP-Arg; Robinson et al., 2011) as a control marker. Duodenal digesta was collected every 2 or 3 h for 48 – 54 h and was homogenized to extract free Lys and Arg. Changes of Lys and Arg concentrations in digesta were plotted to calculate the area under the curve (AUC). The proportion of AUC of Lys to Arg was defined as the bypass rate of AjiPro-L. To determine the fecal excretion, feces from each cow were collected for 72 h after ingestion of AjiPro-L. Feces were homogenized with hot water to extract free Lys from the products. Amounts of Lys excreted in feces were calculated by analyzing free Lys concentration in the extract. Statistical differences were tested by an ANOVA. In situ ruminal protection of A2G was lower (P < 0.01) than A1G (97 vs. 99% at 6 h, 94 vs 97% at 12 h and 84 vs 90% at 24 h, respectively), but still reasonably high. The bypass rate of Lys from A2G (75 ± 11%) was not different (P = 0.45) from A1G (82 ± 14%), but fecal excreted Lys from A2G (33 ± 6%) was significantly less (P < 0.01) than A1G (51 ± 1%). Our results indicate that higher bioavailability of A2G than A1G was attributed to improved intestinal digestibility, supporting the results obtained by the in vivo plasma Lys response method.