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Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4~ in a stabilization solution (60). These extracts gelled on warming to 25~ and contracted in response to micromolar Ca ++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis(/3-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis[2-ethane sulfonic acid] (PIPES), 1 mM MgCI2, 1 mM ATP, and 20 mM KCI at pH 7.0 (relaxation solution), while micromolar levels of Ca ++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca ++, pH >7.0, cytochalasin B, elevated concentrations of KC1, MgCl2, and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca ++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material squeezed out the contracting extract; (c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4-to 10-fold.In the absence of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absence of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding purified Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++-regulated gelation.These results indicated: (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel,

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