Class I β-1,3-glucanase (βGLU I) is transcriptionally induced in the micropylar endosperm just before its rupture prior to the germination (i.e. radicle emergence) of Nicotiana tabacum L. cv. ‘Havana 425’ seeds. Ethylene is involved in endosperm rupture and high-level βGLU I expression; but, it does not affect the spatial and temporal pattern of βGLU I expression. A promoter deletion analysis of the tobacco βGLU I B gene suggests that (1) the distal −1452 to −1193 region, which contains the positively acting ethylene-responsive element (ERE), is required for high-level, ethylene-sensitive expression, (2) the regions −1452 to −1193 and −402 to 0 contribute to down-regulation by abscisic acid (ABA), and (3) the region −402 to −211 is necessary and sufficient for low-level micropylar-endosperm-specific expression. Transcripts of the ERE-binding proteins (EREBPs) showed a novel pattern of expression during seed germination: light or gibberellin was required for EREBP-3 and EREBP-4 expression; EREBP-4 expression was constitutive and unaffected by ABA or ethylene; EREBP-3 showed transient induction just before endosperm rupture, which was earlier in ethylene-treated seeds and inhibited by ABA. No expression of EREBP-1 and EREBP-2 was detected. In contrast to βGLU I, EREBP-3 and EREBP-4 were not expressed specifically in the micropylar endosperm. The results suggest that transcriptional regulation of βGLU I could depend on: activation of ethylene signalling pathways acting via EREBP-3 with the ERE as the target, and ethylene-independent signalling pathways with targets in the proximal promoter region that are likely to determine spatial and temporal patterns of expression.