Y (immune) interferon (IFN-'y) was induced in cultures of fresh human lymphocytes by combined treatment with a phorbol ester (12-0-tetradecanoylphorbol 13-acetate, TPA) and the T cell mitogen phytohemagglutinin (PHA). Compared to the IFN-y yields obtained with PHA induction alone, the inclusion of TPA caused a significant enhancement of IFN-y production.Poly(A)-containing mRNA was isolated from mononuclear cells induced with TPA and PHA. Injected into Xenopus laevis oocytes, this mRNA preparation gave rise to IFN activity with characteristic properties of human IFN-y. Sucrose density gradient centrifugation analysis showed that IFN-y mRNA sedimented at 15 S, suggesting that it contains a total of about 1400 nucleotides.As a result ofrecent progress in the study ofvarious interferons (IFNs), all known IFNs can be grouped into three major classes designated IFN-a ("leukocyte"), 3 ("fibroblast"), and IFN-'y ("immune"). Identification of the three major IFN classes is based primarily on antigenic differences (1). Additional distinguishing properties include differences in various physicochemical and biological characteristics (2).Several earlier studies showed that mRNA for IFN-a and IFN-f3 can be extracted from cells actively producing IFN. Such IFN mRNA preparations can direct synthesis of biologically active IFN molecules with corresponding specificity when introduced into heterologous animal cells (3), suitable cell-free systems (4-6), or Xenopus laevis oocytes (6-8). Identification of human IFN-a and IFN-,B mRNAs on the basis of their ability to direct the synthesis of homologous, biologically active IFN molecules on injection in X. laevis oocytes made possible the preparation of cDNAs and their cloning in bacteria (9, 10). Comparison of the first available IFN-a and IFN-/3 cDNA sequences indicated only 29% homology between the two IFNs at the amino acid level (11). Recent analysis of human chromosomal gene bank DNA revealed the existence ofat least eight related but distinct IFN-a sequences, differing from each other in about 10-30% of their predicted amino acid residues (12). In contrast, only one subclass ofIFN-,BmRNA has been tentatively identified in addition to the IFN-,f cDNA clone originally isolated (13).Studies of isolated IFN-a and IFN-fB DNA clones and of the corresponding IFN polypeptides produced in bacteria have already provided a wealth ofinformation about the structure and biological properties ofthese IFNs. In addition, cloning ofthese IFN genes in Escherichia coli promises to facilitate the production of large quantities of biologically active IFN.In contrast, much less information is available about IFN-y. The latter IFN is usually produced in cultures of lymphocytes exposed to various mitogenic stimuli-e.g., specific antigens or lectins. Recently we described an efficient method of production and partial purification ofhuman . In this paper we report on the preparation ofbiologically active human IFN-'y mRNA. It is hoped that the preparation of IFN-y mRNA will allow the synthesis ...