ABSTRACTBiochemical studies of human actin and its binding partners rely heavily on abundant and easily purified α-actin from skeletal muscle. Therefore, muscle actin has been used to evaluate and determine the activities of most actin regulatory proteins but there is an underlying concern that these proteins perform differently from actin present in non-muscle cells. To provide easily accessible and relatively abundant sources of human β- or γ-actin (i.e. cytoplasmic actins), we developedSaccharomyces cerevisiaestrains that express each as their sole source of actin. Both β- or γ-actin purified in this system polymerize and interact with various binding partners, including profilin, mDia1 (formin), fascin and thymosin-β4 (Tβ4). Notably, Tβ4 and profilin bind to β- or γ-actin with higher affinity than to α-actin, emphasizing the value of testing actin ligands with specific actin isoforms. These reagents will make specific isoforms of actin more accessible for future studies on actin regulation.