Barley has a low glycemic index (GI), but it is unknown whether its GI is affected by variation in carbohydrate composition in different cultivars and by food processing and food form. To examine the effect of these factors on GI, 9 barley cultivars varying in amylose and β-glucan content were studied in 3 experiments in separate groups of 10 healthy participants. In Expt. 1, 3 barley cultivars underwent 2 levels of processing: hull removal [whole-grain (WG)] and bran, germ, and crease removal [white pearled (WP)]. GI varied by cultivar (CDC Fibar vs. AC Parkhill, [mean ± SEM]: 26 ± 3 vs. 53 ± 4, respectively; P < 0.05) and pearling (WG vs. WP: 26 ± 4 vs. 35 ± 3, respectively; P < 0.05) with no cultivar × pearling interaction. In Expt. 2, the GI of 7 WG cultivars ranged from 21 ± 4 to 36 ± 8 (P = 0.09). In Expt. 3, WG and WP AC Parkhill and Celebrity cultivars were ground and made into wet pasta. The GI of AC Parkhill pasta (69 ± 3) was similar to that of Celebrity pasta (64 ± 4) but, unlike in Expt. 1, the GI of WP pasta (61 ± 3) was less than that of WG pasta (72 ± 4) (P < 0.05). Pooled data from Expts. 1 and 2 showed that GI was correlated with total fiber (r = -0.75, P = 0.002) but not with measures of starch characteristics. We conclude that the GI of barley is influenced by cultivar, processing, and food form but is not predicted by its content of amylose or other starch characteristics.