Growth of three clinical isolates (Scott A, Murray B, and F5380) and one laboratory strain (EGD) of L. monocytogenes at 4 degrees C significantly increased their virulence for intravenously injected mice. Using the EGD strain for subsequent experiments, we determined that growth at either 4 degrees or 22 degrees C enhanced the growth of listeria in the spleen and liver. Similar numbers of listeriae were recovered from the spleens and livers of mice during the first 48 h after i.v. injection of strain EGD grown at 37 degrees C or 4 degrees C. At later timepoints (3-6 days), significantly more listeriae were recovered from the spleens and livers of mice injected i.v. with strain EGD grown at 4 degrees C. In contrast, L. monocytogenes EGD grown at 37 degrees C and 4 degrees C demonstrated similar abilities to survive in the gastrointestinal tract, to translocate to the mesenteric lymph nodes, and to disseminate to the spleen and liver in intragastrically inoculated mice. Listeria monocytogenes EGD grown at 4 degrees C released less hemolysin into the culture medium than did this strain when grown at 22 degrees C and 37 degrees C. Transfer to fresh broth and incubation at 37 degrees C for 2 h increased the release, to similar levels, of hemolysin from L. monocytogenes EGD grown at 4 degrees, 22 degrees, and 37 degrees C. Temperature-induced differences in virulence, therefore, may not reflect the amount of hemolysin released.