In response to heat shock (34³C, 30 min), cell morphology and actin organization in Dictyostelium discoideum are drastically changed. Loss of pseudopodia and disappearance of F-actin-containing structures were observed by using fluorescence microscopy. These changes were paralleled by a rapid decrease of the F-actin content measured by a TRITC-phalloidin binding assay. The effects of heat shock on cell morphology and actin organization are transient : After heat shock (34³C) or during a long-term heat treatment (30³C), cell morphology, F-actin patterns and F-actin content recovered/adapted to a state which is characteristic for untreated cells. Because F-actin may be stabilized by increased amounts of heat shock proteins, their response and interaction with F-actin was analyzed. After a 1 h heat treatment (34³C), the major heat shock protein of D. discoideum (HSP70) showed maximally increased synthesis rates and levels. During recovery from a 34³C shock or during a continuous heat treatment at 30³C, the HSP70 content first increased and then declined slowly toward normal levels. Pre-treatment of cells with a short heat shock of 30 min at 34³C stabilized the F-actin content when the cells were exposed to a second heat shock. Furthermore, a transient colocalization of HSP70 and actin was observed at the beginning of heat treatment (30³C) using immunological detection of HSP70 in the cytoskeletal actin fraction. ß