We devised a micro-suspension-test to evaluate disinfectants against human immunodeficiency virus type-1 (HIV-1) and confirmed its reliability. Suspensions of persistently HIV-1-infected Molt-4 cells were used as targets of disinfectants and residual infectivity was measured by an infectivity assay: after cocultivation with uninfected Molt-4 cells reverse transcriptase activity (RTA) in the supernatant and giant cell formation (GCF) were monitored. Our new infectivity assay consists of a short-term assay, that is RTA and GCF monitoring on the second day of co-culture, and a long-term assay, that is RTA monitoring up to the 28th day of co-culture. The sensitivity of the short-term assay was 1 x 10(3) infected cells and that of the long-term assay 1 x 10(1) infected cells. All the chemical disinfectants examined in this study showed dose- and time-dependent inactivation of HIV-1. By 5-minute contact with ethanol, glutaraldehyde, formalin, sodium hypochlorite and povidone-iodine, HIV-1 was effectively inactivated at concentrations of 20, 0.01, 5, 0.05 and 0.1%, respectively. Since the micro-suspension-test is easy and sensitive, we recommend it as a method for evaluating disinfectants against HIV-1.