Expression of six Hsp70s in spinach (Spinacia oleracea cv Longstanding Bloomsdale) leaves grown under isothermal conditions is regulated by a light/dark (L/D) mechanism distinctly different from the light-regulated mechanism for the chlorophyll a/b-binding protein (cab) or small subunit of ribulose-1,5-bisphosphate carboxylase oxygenase (rbcS). Subjecting entrained plants to two or three L/D cycles within a 24-h period resulted in an equal number of oscillations in expression for five out of six 70-kD heat shock proteins (Hsp70s). Three cycles appear to be the maximum, as shorter L/D treatments do not consistently increase the number of cycles in a 24-h period. The expression response of Hsp70s to L/D is overridden by heat shock. Protein disulfide isomerase, a second molecular chaperone of the endoplasmic reticulum, has an expression pattern in entrained plants that is similar to hsc70-2, the endoplasmic reticulum luminal Hsp70 binding protein. The parallel expression patterns for the various Hsp70s and protein disulfide isomerase indicate a likely general coordinate L/D regulation for molecular chaperones in plants. Multiple inductions in response to successive L/D treatments within a 24-h period in entrained plants for five of six Hsp70s support the conclusion that expression is not a consequence of circadian control, but instead is independently cued by non-circadian-mediated L/D signals where peak Hsp70 expression precedes the daily thermoperiod maximum.