(1) Our radioimmunoassay for ubiquitin -- a small, highly conserved "signal" molecule involved in ATP-dependent intracellular proteolysis -- recognizes only free ubiquitin. To measure total ubiquitin (protein bound + free) requires prolonged tissue hydrolysis; its profile is similar to that previously described for the free component i.e. highest titers in egg and adults, and lowest in larvae and pupae. Pure blowfly ubiquitin has been isolated and partially characterized. The ATP-ubiquitin proteolytic system from either the blowfly or from rabbit reticulocytes does hydrolyse the blowfly storage protein calliphorin. (2) Enriched lysosomal preparations from late pharate adult flies very actively hydrolyse bovine serum albumin and denatured hemoglobin; but they attack calliphorin at only 1-2% of the rate for the unnatural substrates.