The conformational analysis of the backbone angle gamma (O5’-C5’-C4’-C3’) and the stereospecific assignment of the H5’((pro-S)), H5’((pro-R)) diastereotopic protons in a uniformly C-13,N-15 labeled RNA oligonucleotide was performed using new Exclusive COSY (E.COSY) type multidimensional heteronuclear NMR experiments designed to measure (3)J(H4’,H5’) homonuclear and (2)J(C4’,H5’ heteronuclear coupling constants. The experiments were demonstrated on a uniformly C-13,N-15 labeled 19-mer RNA hairpin (5’rGCACCGUUGGUAGCGGUGC-3’) derived from the RNA I transcript involved in Col El replication control. From the small (3)J(H4’,H5’) couplings constants observed for the RNA hairpin, it was concluded that all gamma angles assume a gauche(+) rotamer conformation (gamma = 60 degrees). From the signs of the (2)J(C4’,H5’) coupling constants, the H5’ protons were stereospecifically assigned. In the helical stem region of the hairpin, the H5’((pro-S)) protons were found to resonate downfield (similar to 0.4 ppm) of the H5’((pro-R)) protons. In the loop region of the hairpin, the chemical shift differences between the H5’((pro-S)), H5’((pro-R)) resonances were found to be smaller, and in most cases, the H5’((pro-S)) protons were found to resonate upfield of the H5’((pro-R)) protons. The different chemical shift patterns observed for the H5’((pro-R)) and H5’((pro-R)) protons in the two secondary structure elements are discussed.