Using [14C]adenine as a labeled precursor, the biosynthesis and processing of heterogeneous nuclear RNA (hnRNA) in chromatin and nucleoplasm were studied. It was shown that intraperitoneally injected actinomycin D (50-500 micrograms/100 g of body weight) did not inhibit the biosynthesis of hnRNA and rRNA in rat thymocytes. Besides biosynthesis, the polyadenylation of the bulk of primary transcripts was also localized in the chromatin. rRNA precursors constituted only a small part of the newly synthesized hnRNA molecules. During processing, highly polymeric (28S-55S) poly(A+)-hnRNA fractions passed from chromatin to the nucleoplasm, as a result of which a large number of high molecular weight polyadenylated hnRNA molecular were pooled in the nucleoplasm. Thymocyte hnRNA was tightly bound to nuclear structures and its isolation tributed stringent conditions. The bulk of poly(A+)-hnRNA molecules were degraded without formation of stable intermediates. The pattern of poly(A-)-hnRNA maturation in chromatin and nucleoplasm was identical, i.e., in both compartments the decrease in the size and accumulation of processed 16S-20S RNA molecules was observed. About 6% of newly synthesized poly(A+)-hnRNA was converted into polysomal mRNA. Cytoplasmic poly(A+)-RNA consists of two fractions, i.e., metabolically stable high molecular weight (greater than or equal to 40S) RNA molecules and a less stable 10S-20S RNA.