To evaluate the hypothesis that the 3' poly(A) tract of mRNA plays a role in translational initiation, we constructed derivatives of pSP65 which direct the in vitro synthesis of mRNAs with different poly(A) tail lengths and compared, in reticulocyte extracts, the relative efficiencies with which such mRNAs were translated, degraded, recruited into polysomes, and assembled into messenger ribonucleoproteins or intermediates in the translational initiation pathway. Relative to mRNAs which were (14,19,39,48,50). These poly(A) "tails" are added post-transcriptionally in the nucleus, with an initial length, in mammalian cells, of approximately 200 to 250 adenylate residues (9). Following transport of mRNA to the cytoplasm, poly(A) tracts are gradually shortened so that, in steady state, poly(A) tail lengths are heterogeneous, ranging from 50 to 70 A's (2,8,10,38,59,80). Although it has been almost two decades since the discovery of these poly(A) tracts, their function(s) has yet to be clarified.Earlier results from our laboratory (37, 53, 54, 61, 79) led us to propose that poly(A) has a role in translation. More specifically, we proposed that an interaction of the cytoplasmic poly(A)-binding protein (PABP) with a critical minimum length of poly(A) facilitates the initiation of translation of polyadenylated [poly(A)+] but not nonpolyadenylated [poly(A)-] mRNAs. The results of several different experimental approaches have provided evidence which indirectly supports this hypothesis. These results include (i) the correlation of specific changes in mRNA poly(A) tail length with translational efficiency following fertilization in Spisula oocytes (72), during early development in Dictyostelium discoideum (61), oocyte maturation in Xenopus laevis (34,55), oocyte activation in mice (33,84,86), salivary gland development in Drosophila melanogaster (67), and in response to physiologic stimuli in the rat hypothalamus (11,69,89); (ii) the higher translational activity of poly(A)+ as opposed to poly(A)-mRNAs in vitro, as demonstrated in systems as diverse as cell extracts derived from rabbit reticulocytes (16, 73), Ehrlich ascites tumor cells (31), and wheat germ (73), as well as in microinjected Xenopus oocytes (15,21,34) and electroporated tobacco protoplasts (22); (iii) a correlation between the abundance and stability of PABPs and the rate of translational initiation in developing or heat-shocked Dictyostelium discoideum amoebae (53,