Rodent brain has been reported to contain a fraction of non-polyadenylated (poly(A)-) mRNA that includes about 100,000 different sequences, most of which are not found in the poly(A)+ fraction. We have prepared a cDNA library of low-abundance poly(A)- RNAs from rat brain polysomes, and have characterized three clones in detail. Two of the clones hybridize on Northern blots to poly(A)+ RNAs from brain. Dot blot hybridization and RNase protection assays demonstrate that although the bulk of the RNA complementary to these clones is present in the poly(A)- fraction, a small portion (7-21%) is present in the poly(A)+ fraction. Our results suggest that the poly(A)-mRNA fraction from rat brain may not contain sequences that are different from those in the poly(A)+ fraction.