The growth pattern of Salmonella enterica subsp. enterica serotype Typhimurium (Typhimurium) was studied in mice to examine the role of the 60-Mdal virulence-associated plasmid in the pathogenesis of mouse typhoid. After repeated subcultures at 45 degrees C, isogenic variants harbouring the virulence-associated plasmid (strains C52, TM122 and TM332) or having lost this large plasmid (strains C53, TM123 and TM333) were obtained from three parental strains (strains C5, TM12 and TM33, respectively). Plasmid pIP1350, present in strain C52, was tagged by Tn10 and transferred by successive conjugations to strains C53, TM123 and TM333. The behaviour of these three Typhimurium lines was studied in C57BL/6, DBA2, B6D2 (C57BL/6 X DBA2 F1 hybrid) and OF1 mice after oral infection, subcutaneous injection into the hind footpad or intravenous inoculation. The kinetics of organ colonization were followed at intervals after injection by enumeration of viable bacteria in caecum, mesenteric or popliteal lymph node, spleen, liver, kidney and lung depending on the route of infection. Strains harbouring virulence plasmid and their cured derivatives did not differ significantly in their ability to colonize caecal content and to translocate to draining lymph nodes. Elimination of the virulence plasmid was correlated with a significant reduction in the ability of cured variants to colonize spleen and liver. Reintroduction of the virulence plasmid into plasmidless variants restored the virulence to the level originally observed. These data demonstrate that a 60-Mdal plasmid in Typhimurium strains is necessary to ensure colonization of spleen and liver of experimentally infected mice.