The structure of the mouse neurotrophin-3 (NT-3) gene has been analysed using genomic cloning and the rapid amplification of cDNA ends (RACE) method. The gene consists of two small upstream exons (exons IA and IB) and a larger downstream exon (exon II) that encodes the mature protein. Two classes of NT-3 transcripts, termed transcripts A and B, are generated by alternative splicing of exon IA or exon IB to the common exon II. The NT-3 gene also contains several transcription start sites in both upstream exons, and three different polyadenylation sites in exon II, as shown by RNase protection assays and by RACE, giving rise to multiple NT-3 mRNA variants of slightly different lengths. Cerebellar granule neurons express both classes of NT-3 transcripts, but only transcript B is regulated by tri-iodothyronine (T3) in these neurons. The effect of T3 on NT-3 mRNA is primarily due to transcription enhancement, as shown in nuclear run-on experiments. The levels of NT-3 mRNA are much lower in cultured mouse astrocytes and are undetectable in the human neuroblastoma cell line IMR 32. A TATA box is present in the upstream region of exon IB but not in that of exon IA. Promoter analysis using the chloramphenicol acetyltransferase reporter gene fused to different NT-3 upstream regions showed the presence of two active NT-3 promoters in cerebellar granule neurons. However, in IMR 32 cells, NT-3 promoter activity decreased dramatically with increasing length of the 5' flanking region. This suggests that expression of the NT-3 gene is regulated both by positive influences, such as T3, and by negative silencing elements present in the upstream regions of the NT-3 promoter.