The expression of transcriptionally active nerve growth factor-induced gene-B (NGFI-B) is rapidly induced in thyroid follicular cells in response to cAMP stimulation. As the transcription of thyrocyte-specific genes is controlled by the cAMP cascade, we have investigated a possible involvement of NGFI-B in this control. Recombinant adenoviruses driving the expression of either the intact NGFI-B protein or a truncated form of it that lacks the capacity to transactivate a NBRE-dependent promoter, were used to infect dog thyrocytes maintained in primary culture. Northern blot analysis of total RNA from infected cells revealed that the expression of NGFI-B was not sufficient to induce a significant accumulation of specific transcripts (thyroglobulin, thyroperoxidase, sodium-iodide symporter) in unstimulated thyrocytes. The overproduction of the transcriptionally inactive form of NGFI-B in thyrocytes maintained in the presence of forskolin after infection did not impair the accumulation of the thyroid-specific transcripts. These data show that NGFI-B does not control the expression of differentiation in thyrocytes by acting through a canonical NBRE. As a consequence, we must consider that either the expression of NGFI-B in cAMP-stimulated thyrocytes is not critically linked to the expression of differentiation or that NGFI-B is implicated in a regulatory mechanism which differs from its known action at the level of a NBRE.