It has previously been shown that a far-upstream region of the herpes simplex virus immediate early (IE) gene 3 increases the expression of a linked thymidine kinase (TK) gene and also contains sequences which respond to stimulation of transcription by a virion component. The effects of altering the orientation and distance of the far-upstream region with respect to the normal IE gene 3 promoter are described. Reversal of the orientation whilst retaining the normal position of the far-upstream region did not affect its activity, but placing it downstream from the TK gene abolished any detectable effect. When the far-upstream region was separated from the promoter by insertion of Escherichia coli DNA fragments (approximately 1000 base pairs), its activity was reduced but not prevented. A similar effect was observed for unstimulated expression of TK and stimulated expression in the presence of the virion component. The IE gene 3 far-upstream region therefore resembles enhancer sequences in some respects but also shows significant differences.