Cytoplasmic dynein is a microtubule (MT) motor protein comprising two classes: dynein-1 and dynein-2. We purified recombinant human dynein-1 and dynein-2 from HEK-293 cells by expressing the streptavidin-binding peptide-tagged human cytoplasmic dynein-1 and dynein-2 heavy chains (HCs), respectively. Electron microscopy of the purified molecules revealed a two-headed structure composed of characteristic dynein motor domains. In an in vitro MT gliding assay, both dynein-1 and dynein-2 showed minus-end-directed motor activities. This is the first demonstration of dynein-2 motor activity, which supports the retrograde intraflagellar transport role of dynein-2. Our expression system of dynein HCs provides a useful means to investigate dynein functions. Structured summary of protein interactionsDYNC1H1 (Homo sapiens)binds to DYNC1H1 (Homo sapiens) by electron microscopy(View interaction)DYNC1H1 (Sus scrofa)binds to DYNC1H1 (Sus scrofa) by electron microscopy(View interaction)DYNC2H1 (Homo sapiens)binds to DYNC2H1 (Homo sapiens) by electron microscopy(View interaction)