Eukaryotic cells contain a large number of small nucleolar RNAs (snoRNAs). A major family of snoRNAs features a consensus ACA motif positioned 3 nucleotides from the 3' end of the RNA. In this study we have characterized nine novel human ACA snoRNAs (U64-U72). Structural probing of U64 RNA followed by systematic computer modeling of all known box ACA snoRNAs revealed that this class of snoRNAs is defined by a phylogenetically conserved secondary structure. The ACA snoRNAs fold into two hairpin structures connected by a single-stranded hinge region and followed by a short 3' tail. The hinge region carries an evolutionarily conserved sequence motif, called box H (consensus, AnAnnA). The H box, probably in concert with the flanking helix structures and the ACA box characterized previously, plays an essential role in the accumulation of human U64 intronic snoRNA. The correct processing of a yeast ACA snoRNA, snR36, in mammalian cells demonstrated that the cis- and trans-acting elements required for processing and accumulation of ACA snoRNAs are evolutionarily conserved. The notion that ACA snoRNAs share a common secondary structure and conserved box elements that likely function as binding sites for common proteins (e.g., GAR1) suggests that these RNAs possess closely related nucleolar functions.