Molecular dynamics simulations of RNA microhelix(Ala) indicate that G:U and other 3:70 purine: pyrimidine wobble pairs induce local deviations from A-form geometry in their respective microhelices; the helix is underwound at the base-pair step above and overwound at the base-pair step below, in each case by about 7-9 degrees compared to canonical A-form RNA. On the basis of analysis of average water densities and residence lifetimes, the wild-type microhelix strongly binds a water molecule in the minor groove of the 3:70 base pair, consistent with crystallographic analyses of an RNA duplex derived from the acceptor stem of Escherichia coli tRNA(Ala). Other wobble pairs show water binding at this position but to a lesser degree; the strength of water binding correlates directly with the measured aminoacylation activities of the microhelices as substrates for E. coli alanyl-tRNA synthetase (G:U > 2AA:IsoC > G:dU > I:U). Watson-Crick base pairs at the 3:70 position show no tendency toward specific hydration. This tightly bound minor-groove water in the microhelices with 3:70 wobble pairs evidently does not function to stabilize a particular local helical structure, but it may play a role as a specific recognition element or serve as an indicator of interaction specificity between the microhelix and a hydrogen-bonding residue of the aminoacyl-tRNA synthetase.