Thein vitrotranscriptional activity of the two 6-crystallin genes (5′-61–62–3′) of the chicken was studied in a whole Hela cell extract. Both the 61 and 62 promoters were recognized by RNA polymerase II in this heterologous system. The major RNA initiation site from the 61 promoter was the samein vitroas that which occursin vivo, as judged by mapping with S1-nuclease, although other minor initiation sites upstream and downstream of the major initiation site were noted. A primer extension experiment showed that the longest RNA synthesizedin vitrofrom a 62 template initiated near the beginning of the first exon. The 61 promoter was several-fold stronger than that of 62 under the presentin vitroconditions. Transcription from the $dL1 promoter was abolished by a competitor fragment (c'-II; includes -328 to -63) purified from the 62 promoter, indicating that one or more common transcription factors binding upstream from the TATA box are required forin vitrofunction of the two $dL-crystallin promoters. Thus, in the Hela cell extract both $dL-crystallin genes contain a functional promoter. We consider the possibility that the single 5'CCAAT3′sequence present in the $dL1 promoter (but lacking in the $dL2 promoter) may contribute to its greater core activity under our conditions. The greater promoter activity of the $dL1-crystallin gene in the Hela cell extract was not sufficient to account for the large ratio of $dL1 to $dL2 mRNA (-50 to 100) in the embryonic chicken lens.