In various toxicological studies, single housing of rodents is preferred to standardize for regulatory purposes. However, housing conditions can have severe, often underestimated, impact on results in toxicological examinations. As different husbandry conditions have been shown to impose stress, we investigated their influence on plasma cytokines. Adult male Wistar rats were assigned to one group housed in cages of four and another housed singly for 28 days. Eight animals per group were tested in the forced swim test (FST) for symptoms of "behavioral despair," and in another eight animals per group, plasma concentrations of the stress hormone ACTH, of the pro-inflammatory cytokines TNF-α, IFN-γ, IL-2 and IL-22, and of the anti-inflammatory cytokines IL-4 and IL-10 were analyzed. Group-housed animals had significantly lower body weight than individually housed animals. The FST revealed symptoms of "behavioral despair" of individually housed rats accompanied by higher levels of ACTH and TNF-α but also of IL-4 and IL-10. No significant differences between housing conditions were found for IFN-γ, IL-2 and IL-22. Social isolation by husbandry conditions, apart from any other manipulation, alters the behavioral and immunological status of rats and must be considered when immunological effects are examined in various experimental protocols.