AbstractObjectives were to compare in vitro and in situ disappearance of dry matter (DM), neutral detergent fiber (NDF), and starch of traditional (unprocessed and rolled) and hulless (unprocessed) barley. Experiment 1: three barley sources were compared using in vitro techniques. The sources were: 1) traditional barley that was not processed, 2) traditional barley processed through a roller mill, and 3) hulless barley that was not processed. For in vitro incubation, each barley source was ground through a 1-mm screen. Ground barley sources were weighed into bags (25 micron porosity) and incubated in ruminal fluid from two steers fed 80% rolled corn for 3, 6, 12, 24, 48, or 72 h. Intact bags were assayed for NDF; remaining bags were opened and the residual was removed and analyzed to determine disappearance of DM and starch. Experiment 2: the barley sources used in Exp. 1 were compared using in situ techniques. For in situ analysis, each barley source was ground in a Wiley mill with no screen to mimic mastication. Artificially masticated samples were weighed into Dacron bags (50 ± 10 micron porosity) and incubated in eight ruminally fistulated steers (n = 8) for 3, 6, 12, 24, 48, and 72 h. Residual contents were analyzed to determine in situ disappearance of DM, NDF, and starch. Data were analyzed using the MIXED procedures of SAS (9.4 SAS Institute, Cary, NC) with repeated measures. DM disappearance was greatest (P < 0.05) for hulless barley in vitro and for rolled barley in situ, regardless of time postincubation. For both trials, NDF disappearance was greatest (P < 0.05) for hulless barley, regardless of time postincubation. Starch disappearance at all time points was greatest (P < 0.05) for rolled barley in situ. Starch disappearance was greater (P < 0.05) for hulless barley at 6 h of in vitro incubation compared to rolled and unprocessed barley, whereas starch disappearance in vitro was comparable (P = 0.60) between barley sources. When the grains were compared in vitro, minor differences were noted, presumably because barley sources were finely ground prior to incubation. Compared to in vitro estimates, in situ techniques had greater variation in ruminal degradation estimates. Differences observed between in situ and in vitro techniques are driven largely by differences between the procedures. Although laboratory methods are widely used to estimate ruminal degradation, these techniques did not provide comparable estimates of ruminal degradation of barley.