Splicing of pre-mRNA in HeLa cells exposed to various stress response inducers has been investigated. In vivo, intron-containing transcripts of the hsp27 gene accumulate in cells stressed by heat or sodium arsenite. In vitro analysis, however, reveals a differential effect of stress on splicing: nuclear extracts from cells exposed to a severe heat shock are incapable of splicing an exogenously supplied substrate while splicing is not perturbed in extracts treated with sodium arsenite, the amino acid analog canavinine or ethanol. Pretreatment of cells with a mild heat shock prior to a severe heat shock protects the splicing apparatus and allows splicing to proceed unimpeded. Analyses of the splicing defect in extracts from heat-shocked cells show that the inhibition of splicing cannot be accounted for by changes in the major RNA and protein components of small nuclear ribonucleoprotein particles (snRNPs) or in a previously described heat-labile factor that is essential for in vitro splicing. Fractionation of small nuclear ribonucleoprotein particles from heat-shock extracts by native polyacrylamide gel electrophoresis reveals dramatic changes in certain particles, most noticeably in a U4/U5/U6 snRNP complex and the U2 snRNP. Alterations in these particles are accompanied by the assembly of labeled pre-mRNA transcript into aberrant splicing complexes that differ from those formed in normal extracts.