Previous studies have investigated the relationship between the Spot 14 gene and hepatic lipogenesis. Those studies found that the Spot 14 protein was induced when lipogenesis was induced and suggested that induction of the Spot 14 protein was required for induction of hepatic lipogenesis by thyroid hormone and dietary carbohydrate. Analysis of those findings led us to hypothesize that the Spot 14 gene is required for induced hepatic de novo lipogenesis in vivo. To test this hypothesis, we created an in vivo deletion of the Spot 14 gene in mice using gene-targeting technology. Southern blot analysis showed that the Spot 14 gene was disrupted. Northern blot analysis showed that this disruption ablated expression of intact hepatic Spot 14 mRNA. In contrast to our hypothesis, acute thyroid hormone administration led to comparable induction of hepatic lipogenic enzyme mRNAs between the wild-type and knockout mice. Furthermore, long-term treatment with both thyroid hormone and a diet promoting lipogenesis led to enhanced lipogenic enzyme activity and a greater rate of hepatic de novo lipogenesis in the knockout, compared with the wild-type, mice. Although these data indicate that the Spot 14 protein is not required for induced hepatic de novo lipogenesis, they also suggest that Spot 14 plays some role in this process. It is possible that alternative pathways that complement the loss of the Spot 14 protein are present, and in the absence of Spot 14, these alternative pathways overcompensate to produce an enhanced rate of induced lipogenesis.