The initial day 14 wave of fetal thymocytes express a gamma delta T-cell receptor (TCR). This surface TCR is generated by preferential rearrangement of V gamma 3 and V delta 1 recombination segments. To delineate the role of regulatory sequences in this expression, we have analyzed the V gamma 3 promoter control region under the regulation of its cognate C gamma 1 enhancer. Transcription initiates 25 bases downstream from a TATTAA sequence at a consensus initiator motif. The minimal 5' promoter sequences supporting expression by transient analysis extend -243 nucleotides from the +1 start site. Three regulatory sequences in this region have been defined by deletion and mutagenesis: a consensus CTF/NF-1 site at -55, an Ets homology sequence at -65, and a degenerate, but crucial, SP-1 site at -100. The presence of additional sequences downstream of the start site which extend through the leader intron were necessary for expression. In contrast to other TCR or immunoglobulin variable regions, one or more strong upstream suppressor sequences resembling silencer elements have been observed. A 311-bp fragment, positions -586 to -897, exhibited strong repressing activity regardless of orientation when placed upstream of heterologous promoters.