Heat shock caused significant changes in intracellular pH (pH i ) and intracellular free calcium concentration ( Ca 2+ i) which occurred rapidly after temperature elevation. pH i fell from a resting level value at 25 degrees C of 7.38 +/- 0.02 (mean +/- standard error of the mean, n = 15) to 6.91 +/- 0.11 (n = 7) at 35 degrees C. The resting level value of Ca2+i in single Drosophila melanogaster larval salivary gland cells was 198 +/- 31 nM (n = 4). It increased approximately 10-fold, to 1,870 +/- 770 nM (n = 4), during a heat shock. When salivary glands were incubated in calcium-free, ethylene glycol-bis(beta-aminoethyl ether)-N,N′,N′-tetraacetic acid (EGTA)-buffered medium, the resting level value of Ca 2+ i was reduced to 80 +/- 7 nM (n = 3), and heat shock resulted in a fourfold increase in Ca 2+ i to 353 +/- 90 nM (n = 3). The intracellular free-ion concentrations of Na + , K + , Cl - , and Mg 2+ were 9.6 +/- 0.8, 101.9 +/- 1.7, 36 +/- 1.5, and 2.4 +/- 0.2 mM, respectively, and remained essentially unchanged during a heat shock. Procedures were devised to mimic or block the effects of heat shock on pHi and Ca 2+ i and to assess their role in the induction of heat shock proteins. We report here that the changes in Ca 2+ i and pHi which occur during heat shock are not sufficient, nor are they required, for a complete induction of the heat shock response.