SummaryA library of random yeast genomic DNA:lacZfusions has been constructed using an episomal yeast‐Escherichia colishuttle vector (pCS1). Plasmid pCS1 requires insertion of a promoter and an inframe ATG codon upstream of its resident truncatedlacZgene to regulate expression in yeast. Yeast genomic DNA fragments of 4–6 kb were generated by partial digestion withSau3Aand ligated into the uniqueBamHIsite of plasmid pCS1 to generate a library of 5 × 104individualE. colitransformants. This library was screened to identify promoter‐lacZfusions that were expressed uniquely during sporulation. Of 342 yeast transformants that exhibited β‐galactosidase activity, two were found to express thelacZgene in a sporulation‐specific manner.This paper presents the characterization of two genomic yeast DNA fragments containing promoters that controllacZexpression during the sporulation process. Expression from the promoter present in plasmid pJC18 occurred from 11–21 hours into the sporulation process, while the promoter in plasmid pJC217 was active from 4–14 hours. Staining of nuclear DNA to correlate nuclear morphology with timing of gene expression showed when each of these promoters was active in terms of the morphological stages