AbstractObjectives: Adenovirus 36 (Ad‐36) has been shown to increase adiposity in experimentally infected chickens, mice, and marmosets (nonhuman primates). Neutralizing antibodies to Ad‐36 are associated with obesity in humans. The metabolic and molecular mechanisms responsible for Ad‐36‐induced adipogenesis are unknown. As a potential adipogenic mechanism, this study examined if Ad‐36 enhanced differentiation of preadipocytes.Research Methods and Procedures: To determine the suitability of 3T3‐L1 cells (murine preadipocyte cell line) as a model, the first experiment determined if Ad‐36 attaches and initiates replication in the cells. Next, effects of Ad‐36 on the number of differentiated adipocytes, glycerol 3‐phosphate dehydrogenase (GPDH) levels, and cellular lipid accumulation were determined. The last experiment determined the effect of Ad‐36 on human primary preadipocyte differentiation. Ad‐2, a known nonadipogenic human adenovirus, was used as a negative control in these experiments.Results: Immunofluorescence studies showed adenoviral attachment to 3T3‐L1 cells, and reverse transcriptase‐polymerase chain reaction showed expression of the Ad‐36 E1A gene in the infected cells. Ad‐36, but not Ad‐2, increased the number of differentiated adipocytes, GPDH enzyme levels, and the total cellular lipid content. Also, Ad‐36, but not Ad‐2, increased GPDH levels in human preadipocytes.Discussion: Taken together, these experiments showed that Ad‐36 enhanced differentiation of preadipocytes, which may be a contributory mechanism to its adipogenic effect in vivo. The lack of effect of Ad‐2 on differentiation demonstrated that the observed findings were not a common characteristic of all adenoviruses. Future understanding of the molecular interactions of cellular and viral genes responsible for enhanced differentiation may reveal novel signaling pathways and controls of preadipocyte differentiation.