Locked nucleic acids (LNA, symbols of bases: +A, +C, +G, +T) are introduced into chemically synthesized oligonucleotides to increase duplex stability and specificity. To understand these effects, we have determined thermodynamic parameters of consecutive LNA nucleotides. We present guidelines for design of LNA oligonucleotides and introduce free online software that predicts the stability of any LNA duplex oligomer. Thermodynamic analysis shows that single-strand-to-duplex transition is characterized by the favorable enthalpic change and by the unfavorable loss of entropy. A single LNA modification confines local conformation of nucleotides causing smaller, less unfavorable entropic loss when the single strand is restricted to the rigid duplex structure. Additional LNAs adjacent to the initial modification appear to enhance stacking and H-bonding interactions because they increase the enthalpic contributions towards duplex stabilization. New nearest-neighbor parameters correctly forecast the positive and negative effects of LNAs on mismatch discrimination. Specificity is enhanced in majority of sequences and is dependent on mismatch type and adjacent base pairs; the largest discriminatory boost occurs for the central +C·C mismatch within +T+C+C sequence and the +A·G mismatch within +T+A+G. LNAs do not affect specificity in some sequences, and even impair it for many +G·T and +C·A mismatches. Mismatch discrimination decreases the most for the central +G·T mismatch within +G+G+C and the +C·A mismatch within +G+C+G. We hypothesize that these discrimination changes are not unique features of LNAs but that they originate from the shift of duplex conformation from B-form to A-form.