The objective was to compare the bioavailability of 2 rumen-protected supplements (AjiPro-L 2G vs. AjiPro-L 3G) using the plasma free Lys dose-response technique. Seven lactating multiparous Holstein cows (202 ± 49 DIM) equipped with ruminal cannulas were used in a 7 × 7 Latin square with 7-d periods. The treatments were (1) 0 g/d Lys, (2) 30 g/d of abomasally infused Lys, (3) 60 g/d of abomasally infused Lys, (4) 30 g/d fed Lys from AjiPro-L 2G, (5) 60 g/d fed Lys from AjiPro-L 2G, (6) 30 g/d fed Lys from AjiPro-L 3G, and (7) 60 g/d fed Lys from AjiPro-L 3G. The infusion treatments consisted of Lys-HCl and were infused continuously into the abomasum via the ruminal cannulas. To ensure complete consumption, the AjiPro-L2G and AjiPro-L 3G were mixed with 1.5 kg of TMR, stored at 4°C for 8 h before feeding and placed in tubs in front of the cows 30 min before each of the 3 daily feedings. Blood samples were obtained from each cow on the last 3 d of each period every 2 h, 4 times daily, from the tail vein, centrifuged, deproteinized, composited into 1 daily sample/cow, and analyzed for AA. Data for plasma AA concentrations (μM basis) were analyzed using the PROC MIXED and PROC REG procedures of SAS. Data from 2 cows was removed from the data set due to very low or negative response to all treatments. The slope for the AjiPro-L 3G (i.e., 0.01407; P < 0.001) was greater than the slope for the AjiPro-L 2G (i.e., 0.01257; P < 0.04) resulting in a 12% improvement in bioavailability of Lys from the AjiPro-L 3G based on the ratio of the 2 slopes. Although the bioavailabilities of AjiPro-L 2G and AjiPro-L3G were not significantly different, the 12% numerical increase in bioavailability with feeding AjiPro-L3G results in 1.1 g/d more absorbable Lys when 60 g/d of product are fed.