UV melting curves of mono-and di-2＇-hydroxylated cytosine tetrads were recorded. The substitution of one DNA residue by RNA decreased the melting temperature (T-m) by roughly 6.5 degrees C. In sequences with two RNA residues, strong positional effects were observed. Juxtaposition of the two D-ribose sugars in the tetrad incurred an additional loss of 6 degrees C in tetrad stability and decreased the T-m by 19 degrees C from that of the parent compound, d(TCCCCC)(4). The effect of increasing the number of cytosines on tetrad stability was also measured. Addition (or removal) of one cytosine-protonated cytosine base-pair from the tetrad increased (or decreased) the T-m by roughly; 9 degrees C. Thus, the penalty of adding an RNA residue is equal to or greater than the benefit of the additional base-pair. This implies that the stability of RNA cytosine-tetrads should not rise with increasing length and that thermal transitions previously observed in poly(rC) correspond to duplex structures.