The potential utility of sequentially inoculating a virus sample onto two different cultures of similar vs dissimilar cell lines was evaluated in conjunction with IDU (5-iodo-2'-deoxyuridine) treatment of the cells as a potential adjunct in viral plaque formation assays. This evaluation was done using laboratory grown human echovirus 7, human enterovirus 69 and human poliovirus 1, plus an environmental concentrate derived from sewage that contained indigenous untyped enteroviruses. The cell lines employed were BGM, RD, L-132 and HEL-299. Sequential inoculation generally yielded higher viral assay titers when compared with the more traditional method of simply introducing viral inoculum onto a culture of the first (initial) cell line and then completing the assay without removing that inoculum. When a permissive cell line (BGM or RD) was used for both the initial and final cultures in a sequential inoculation technique, the total plaque count titer from both the initial plus final cultures represented an average 35% improvement over the traditional method.