A heat shock to amebae of the amebo-flagellate Naegleria gruberi during differentiation into swimming flagellates results in the induction of heat shock proteins as well as multiple flagella. The principal heat shock proteins migrate on sodium dodecyl sulfate-polyacrylamide gels with apparent molecular weights of 96,000, 77,000, 70,000, and 68,000. These proteins are synthesized preferentially when cells at 25 degrees C are shifted to temperatures above 32 degrees C. The maximal incorporation of methionine into heat shock proteins occurs at 38.2 degrees C, the temperature at which maximal induction of multiple flagella has been reported. Synthesis of heat shock proteins requires new transcription as judged by the ability of actinomycin D to inhibit their synthesis during the first 15 min of heat shock but not thereafter. Although heat shock can induce multiple flagella only when applied during a restricted interval, heat shock proteins are induced at any time cells are shifted to 38.2 degrees C. The response to heat shock of the Naegleria heat shock proteins resembles that of Drosophila heat shock proteins, but the two groups of proteins differ in both size and number. Naegleria heat shock proteins are, however, strikingly similar in size to a group of heat-induced proteins found in chick embryo fibroblast, mouse L, and BHK cells.