(1) Myosin I heavy chain kinase bound to phospholipid vesicles containing 15-30% acidic phospholipids is maximally activated prior to autophosphorylation, which is also stimulated by the vesicles. Activation is almost entirely due to an increase in Vmax and autophosphorylation involves both intra- and inter-molecular reactions. The domain structure of the kinase has been studied by controlled chymotryptic and tryptic digestion leading to the identification of a strong and a weak phospholipid- (membrane-) and calmodulin-binding site, 3 regions of autophosphorylation and a C-terminal 35-kDa catalytic domain. (2) A 561-base cDNA oligonucleotide, corresponding in sequence to the region from 35-kDa to 11 kDa from the C-terminus, was obtained by RT-PCR. This oligonucleotide was used to screen a cDNA library with 27 positive clones resulting. One of these clones contains the remaining sequence, i.e. to the C-terminus of the kinase, corresponding to the catalytically active C-terminal 35 kDa. (3) The distribution of the phosphorylated (active) forms of the three Acanthamoeba myosin I isozymes was determined by immunoelectron microscopy with antibodies specific for the phosphomyosins. Most interestingly, the concentration of phosphomyosin IC and F-actin were highest in contracting contractile vacuoles, lower in full contractile vacuoles and least in vacuoles that were filling.