Localization of proteins to chloroplasts is thought to be unaffected by heat stress, and this idea has influenced the interpretation of heat stress effects on photosynthesis. Here we show that a nuclear-encoded plastid heat-shock protein (Hsp) accumulated in the cytosol of plants that were grown at 27^⚬C and then heat-stressed at 43^⚬C but accumulated in the chloroplast during heat stress at 37^⚬C. Immunological evidence indicated that other nuclear-encoded chloroplast proteins also accumulated in the cytosol at 43^⚬C but accumulated in the chloroplast at 37^⚬C. Radiolabeling detached leaves with ³⁵S-met confirmed that newly synthesized, nuclear- and chloroplast-encoded proteins accumulated in the chloroplast at 36^⚬C, while at 46^⚬C the chloroplast-encoded proteins accumulated in the chloroplast, but the nuclear-encoded proteins (including Hsps) accumulated in the cytosol. Cytosolic accumulation of nuclear-encoded chloroplast proteins was readily reversible (e.g., within 1-3 h) on return to normal growth temperatures and was observed in evolutionarily and functionally diverse Anthophyta species (C₃, C₄, CAM, monocot, and dicot). These results are the first solid evidence that newly synthesized nuclear-encoded chloroplast proteins accumulate in the cytosol (i.e., localization is disrupted) during severe, but not moderate, heat stress. This has important implications for understanding the effects of heat stress on photosynthesis and may be relevant to other stresses or to proteins localized to other organelles.