Evidence is presented that a homogeneous cytoplasmic species known as 7S RNA is the only abundant RNA in uninfected HeLa cells which can form strong hybrids with the dominant family of middle repetitive DNA sequences in the human genome. These DNA sequences are known collectively as the Alu family, because most of them share a common Alu I restriction site. When purified 7S RNA was hybridized to three different genomic clones containing Alu family DNA sequences, a specific region (or regions) comprising at most half the RNA sequence was protected from mild digestion with T1 ribonuclease; moreover, the hybrids between 7S RNA and cloned Alu family DNA wer imperfect, since T1 RNAase was able to nick the protected 7S RNA sequences under conditions where a true RNA: DNA duplex would have been resistant. This suggests that 7S RNA is encoded either by a small subset of the 300,000 Alu family sequences in the human genome or by an entirely different family of genes. The sequence of 7S RNA has been highly conserved through recent evolution, and in both avian and murine cells the RNA is selectively incorporated into oncornavirus particles during productive infection. The cellular function of 7S RNA is unknown.