Alpha-1 tubulin is the principal alpha-tubulin isotype found in the flagella of the unicellular green alga, Chlamydomonas reinhardii. Although the pattern of tubulin mRNA accumulation and utilization has been examined in some detail in Chlamydomonas (Lefebvre and Rosenbaum 1986), the transcriptional mechanisms establishing tubulin mRNA levels are not understood. To begin an analysis of the alpha-1 tubulin gene transcriptional control elements, we studied a number of promoter mutants of this gene from Chlamydomonas. These mutants, assayed by injection into Xenopus oocyte nuclei, delimit the promoter to 36 bp of DNA upstream of the cap site and 73 bp of the untranslated mRNA leader. A major rate-controlling element lies in a short GC-rich sequence positioned between the TATA homology and the mRNA cap site (position + 1). A similar sequence motif has been found in the same position upstream of all four tubulin genes of Chlamydomonas (Brunke et al. 1984). A 10 bp linker insertion within this sequence abolishes transcription. A far upstream sequence, located in a fragment between -400 and -800, is an efficiency element, whose deletion inhibits transcription in vivo by about 30%. The upstream element (ue) also has the unique ability to drive RNA polymerase II (RNAPII) transcription in vivo when isolated from all downstream promoter elements, unlike any control element described to date. These results suggest that a sequence within the upstream element is an entry site for RNAPII into the tubulin transcription unit.