The in vitro glycation strategy is a powerful approach used in the studies of the sugar-derived posttranslational modifications of the organic bone matrix. To better understand some aspects of bone matrix glycation, we used glucose (glucosylation) or ribose (ribosylation) to modify organic matrix of cortical and cancellous bone originating from human tibias. Both glucosylation and ribosylation led to the formation of higher levels of AGEs and pentosidine (PEN) in cancellous than cortical bone originating from all tested donors (young, middle-age and elderly men and women). More efficient glycation of bone matrix proteins in cancellous bone most likely depended on the higher porosity of this tissue, which facilitated better accessibility of the sugars to the matrix proteins. Interestingly, the levels of PEN formation differed pronouncedly between glucosylation and ribosylation. Ribosylation generated very high levels of PEN (approx. 6-vs. 2.5-fold higher PEN level than in glucosylated samples). Our results suggest that in vitro glycation of bone using glucose leads to the formation of lower levels of AGEs including PEN, whereas ribosylation appears to support a pathway toward PEN formation.