The mechanism of in vitro inactivation of cell-free human immunodeficiency virus (CFHIV) with ascorbic acid (M) or Congo red (CR) was investigated with specific regard to the impact of an excess of magnesium ions on the viral inactivation. Quadruplicate reaction mixtures containing CFHIV were mixed with a virus-inactivating dose of 500 micrograms/ml ascorbic acid in RPMI medium devoid of fetal bovine serum and incubated for 3 h at 4 degrees C in two parallel sets of experiments. AA-free CFHIV and virion-free AA were included in each experiment as the positive and negative controls, respectively. After adding 10(6) MT2 cells to capture the surviving virons, the mixtures were incubated for 1 h at 37 degrees C. The cells from the first set were washed three times with Hanks balanced salt solution (HBSS) only, and those from the second set were washed with HBSS fortified with MgCl2 (1.0 mg/ml). Similarly, inactivation of CFHIV by increasing amounts of CR ranging between 12.5-100 micrograms/ml was also tested for the effect of MgCl2, except that (i) the assay was performed in subdued light, (ii) CFHIV-CR mixtures were incubated at 37 degrees C for 1 h in the dark and (iii) H9 cells were used instead of the MT-2 cells to capture the surviving virions in the test mixtures. The cells were cultured in RPMI with 20% FBS for 5 days at 37 degrees C. The absence of p24 antigen in the culture supernatant of MT2 or H9 cells indicated HIV inactivation by AA or CR, respectively. Remarkably, the cultured cells that were washed with HBSS + MgCl2 consistently expressed p24 antigen at levels comparable with those from the untreated virus control. Therefore, the apparent in vitro inactivation of CFHIV by either AA or CR was reversible as validated by washing of the cells with HBSS + MgCl2 following capture of the virions from CFHIV-AA or CFHIV-CR inactivation mixtures. These observations underscore the need for including extra magnesium ions as a control in validating various protocols used for assessing the in vitro virucidal activity of reverse transcriptase inhibitors, membrane binding dyes, or other candidate chemical agents.