In previous experiments (Palatnik et al., 1979) we used thermal elution from poly(U)-Sepharose to separate messenger RNA from Dictyostelium discoideum into several fractions differing in the length of their poly(A) tracts. In vitro translation of these fractionated RNAs demonstrated that some cellular mRNAs are relatively more abundant in those fractions with long poly(A) tracts (110 to 115 nucleotides) and other cellular mRNAs are more abundant in those fractions with short poly(A) tracts (60 to 65 nucleotides). Our previous experiments also showed that most mRNAs of vegetatively growing cells are synthesized with long poly(A) tracts which shorten, with age in the cell, to a steady-state size of 60 to 65 nucleotides. The experiments of this paper examined the possibility that the mRNAs enriched, respectively, in the long and short poly(A) fractions, represented different stability classes of mRNA and also reflected the involvement of poly(A) in determining mRNA stability. Vegetatively growing cells of Dictyostelium discoideum were incubated with actinomycin D at a concentration which totally inhibited synthesis of RNA. After 18 hours, total cellular RNA was extracted and translated in a messenger RNA-dependent reticulocyte lysate. In vitro translation products were analyzed by two-dimensional polyacrylamide gel electrophoresis and electrophoretic patterns were compared with those obtained from similar, previous analyses of mRNAs of different poly(A) content. Our results showed that, after actinomycin treatment: (a) the translational complexity of the mRNA population decreased substantially; (b) at least 66 major mRNAs either increased or decreased significantly in relative abundance; (c) most of the mRNAs which increased in relative abundance were those which, in untreated cells, are most abundant in the short poly(A) fractions; and (d) most of the mRNAs which decreased in relative abundance were those which, in untreated cells, are most abundant in the long poly(A) fractions. These experiments, coupled with our previous results, lead us to suggest that those mRNAs which are enriched in fractions containing short, steady-state size poly(A) tracts are most stable. Our data do not support models in which poly(A) length regulates mRNA stability. Additional experiments in this paper demonstrate that: (a) a previously identified (Palatnik et al., 1979) class of mRNA which contains only oligo(A) tracts (approx. 25 nucleotides) is also relatively unstable in the presence of actinomycin D; and (b) a major change in the distribution of translatable mRNAs occurs after cells are removed from prolonged exposure to actinomycin D. Many mRNAs which had been substantially reduced by actinomycin D treatment, increased m relative abundance after removal of the drug. Moreover, a large fraction of the mRNAs which had accumulated to very high levels in actinomycin D-treated cells, decreased dramatically in relative abundance after the drug was removed.