One of the factors regulating the accessibility of specific DNA sequences to endonuclease nicking during meiosis is a group of small nuclear RNA molecules, 125 nucleotides in length and transcribed by RNA polymerase III. These molecules (referred to as PsnRNA) are synthesized during meiotic prophase, when chromosomes are undergoing homologous pairing or are already paired. Accessibility to the meiotically active DNA sequences (P DNA) depends on an as-yet-undefined alteration in chromatin structure. PsnRNA is a critical factor in the alteration; it cannot be replaced by other forms of RNA. The specificity of the chromatin sites altered by PsnRNA appears to be a function of sequence complementarity between it and P DNA. Under in vivo conditions the effective action of PsnRNA depends on homologous chromosome pairing. Chromatin sites housing P-DNA sequences in nuclei isolated from cells lacking homologous pairing are not affected by meiotic endonuclease or DNAse II. Accessibility to these sites can be effected by incubation of the nuclei with PsnRNA, but only if the nuclei are from zygotene-pachytene cells. Analyses of pachytene nuclei preincubated with PsnRNA indicate that PsnRNA renders chromatin accessible to at least two endonucleases, meiotic endonuclease and DNAase II, and that it also limits such accessibility to regions housing the complementary P-DNA sequences.