Soluble sugars are thought to play an important role in the fermentation processes of the rumen but their actual fermentation rate has not been fully assessed. Some sugars are also used as markers to assess gut permeability in monogastrics but their use in ruminants can be compromised by the hydrolytic activity of rumen microflora. This study aimed to evaluate the fermentability of some naturally occurring and synthetic soluble sugars. The synthetic soluble sugars were included to verify their possible use as markers for studies of gut permeability in ruminants. In vitro gas and volatile fatty acid (VFA) production from glucose, fructose, xylose, galactose, sucrose, lactose, arabinose mannitol, lactulose and sucralose were measured in a 24 h-incubation trial using ruminal fluid from heifers adapted or not-adapted to additional sugars in the diet, and with caecal content as inocula. Gas production from the same sugars was further evaluated in a 72 h-incubation trial with not-adapted rumen fluid only. Gas and VFA production were not affected by feeding additional sugars, but significant effects of inocula (ruminal vs caecal), sugars and their interaction were observed. Caecal inoculum produced less gas but higher VFA than ruminal inocula. Fructose and glucose had the highest rates of gas production (10.57% h-1 and 10.42% h-1, respectively), and lactulose and mannitol the lowest (3.47% h-1 and 4.63% h-1, respectively) when fermented with ruminal fluid. Sucralose seemed to have a negative effect on microbial fermentations. Our results indicate that lactulose and mannitol might largely escape rumen fermentation, suggesting their possible use as markers to test gut permeability also in ruminants. This needs to be verified in vivo.