The aim of this study was to evaluate the effects rumen outflow in dairy cows fed unsaturated fatty acid by use of reticular and omasal sampling as an alternative to sampling from the abomasal canal. Four Holstein dry cows cannulated in the rumen and abomasum (602 ± 21 kg of body weight; mean ± SD) were assigned randomly into a 4 × 4 Latin square design experiment, fed the following diets: (1) control (C), without unsaturated fatty acid supplementation; (2) soybean oil (SO), addition of 3% of refined soybean oil in DM basis of total diet; (3) whole raw soybean (WS), addition of 16% of whole raw soybeans in DM basis of total diet; and (4) calcium salts of unsaturated fatty acids (CSFA), addition of 3% of CSFA in DM basis of total diet. Nutrient flow was calculated using the reconstitution system based on 3 markers (cobalt-EDTA, ytterbium chloride and indigestible NDF). Large and small particles and the fluid phase were recovered from digesta collected of different sites. Samples were collected 8 times during 20 4 h and were combined into one pool sample for further reconstitution. Data were analyzed using PROC MIXED of SAS 9.1 using the Tukey test to test pair wise comparisons. The DM flow was higher (P < 0.01) to abomasum compared with reticulum and omasum (4.0; 4.0 and 4.8 kg to sites reticulum, omasum and abomasum respectively). However, the NDF flow was higher (P < 0.01) (1.5; 1.2 and 1.4 kg to reticulum, omaso and abomasums respectively), to reticulum compared with omaso and abomasums. There was no difference for the 18:3 flow. The 18:2 flow was higher (50.9; 10.3 and 24.3 g to sites reticulum, omasum and abomasum respectively), to reticulum compared with omaso and abomasums, and 18:1 flow was less (P < 0.01) for to omasum compared with reticulum and abomasum. The reticular sampling technique provided reliable estimates for DM flow and fatty unsaturated fatty acids in dairy cows