The N-acetyl-l-Met (NALM) is a Met derivative produced via protection of l-Met α-amino group with an N-acetyl group. The NALM has been shown to be bioavailable and capable of replacing the dietary requirement for Met in animals and humans. The efficacy of the NALM to provide a source of Met depends on its resistance to microbial degradation in the rumen, its rapid escape from the rumen with digesta, and its subsequent absorption and metabolism to Met in the small intestine. The objective of this study was to quantify the gastrointestinal availability of the NALM by measuring the ruminal escape and intestinal appearance in lactating dairy cows. The current experiment was conducted as a 3 × 3 Latin square design using 3 lactating dairy cows with cannula in the rumen and duodenum. Each period consisted of 10-d adaptation followed by 2 d of sampling. A dose of 0 (control), 30, and 60 g/d of NALM in powder form was placed under the rumen raft at the time of feeding every day. On the first day of sampling, a liquid phase marker (Co-EDTA) was also administered as a bolus dose into the rumen at the time of administration of the NALM. Blood and ruminal and duodenal contents were then taken at hours 0, 1, 3, 6, 9, 12, and 24 post-feeding. A nonlinear regression model was used to determine fractional rate constants for the passage of the liquid marker and the disappearance of NALM from the rumen. There was no difference between treatments for liquid passage and ruminal NALM passage rate. Ruminal escape as a percentage of the dose was numerically higher for the low dose (69.1%) than the high dose (46.2%), but no statistical difference was found (P = 0.44). Duodenal NALM appearance as a percentage of the dose did not differ (P = 0.21) between the low (2.16%) vs. the high dose (3.40%). In addition, NALM treatments at 30 and 60 g/d did not affect ruminal fermentation profiles. In conclusion, overall results from the current study suggest that NALM was effectively protected against microbial degradation in the rumen, particularly at the low dose and extensively digested in the small intestine of lactating dairy cows.