This study evaluated the effect of 2% chlorhexidine (CHX) pretreatment of dentin on the immediate and aged microtensile bond strength (mu TBS) of different adhesives to dentin in vivo and in vitro. Class I cavities were prepared in 80 caries-free human third molars of 40 patients in a split-mouth fashion. In each tooth pair, one tooth received 2% CHX pretreatment after which both teeth were randomly assigned to one of the following groups with respect to the type of adhesive system applied: Adper Single Bond 2 (etch-and-rinse), Clearfil SE Bond (two-step self-etch), Clearfil S-3 Bond (one-step self-etch), and Adper Prompt-L-Pop (all-in-one self-etch). The teeth were restored with resin composite and extracted for mu TBS testing either immediately or after six months in function. In vitro specimen pairs were prepared as with the clinical protocol in intact, freshly extracted human molars, and thereafter, subjected to testing immediately or after 50003 thermocycling. Data were analyzed with four-way analysis of variance (ANOVA). Bonferroni test was utilized for pair-wise comparisons. The immediate bond strength values were significantly higher than "aged'' ones for all tested adhesives (p=0.00). The in vitro immediate bond strength values were statistically higher than in vivo bond strength values (p<.05). While the bond strength of in vitro aged, CHX-treated samples were higher than their in vivo counterparts (p<.05), no difference was observed in non-CHX treated groups (p>0.05). In the absence of CHX pretreatment, all adhesives showed significantly higher immediate bond strength values than CHX-treated groups, while all "aged'', non-pretreated adhesives exhibited significantly lower bond strength values (both p<0.05). By contrast, chlorhexidine pretreatment resulted in significantly higher aged bond strengths, regardless of the adhesive system and testing condition. Aging-associated decline in dentin bond strength of etchand rinse and self-etch adhesives can be counteracted by chlorhexidine application.