To determine whether changes in gene expression occur in embryonic cells as a consequence of changes in cellular aggregation, chicken embryo brain (CEB) cells isolated from 8-day embryos were allowed to aggregate or prevented from aggregating by treatment with anti-neural cell adhesion molecule (N-CAM) Fab' fragments. A subtractive hybridization cloning strategy was employed to identify genes that might show different levels of expression in the two populations of cells. In addition, the transcription rates of a number of genes specifying CAMs and transcription factors were directly estimated by using nuclear run-off transcription assays. The transcription rates of several genes, including those encoding N-CAM, Ng-CAM, alpha-N-catenin, HoxA4 (Hox1.4), a fatty acid-binding protein, and a subunit of the mitochondrially encoded cytochrome-c oxidase enzyme decreased upon CEB cell aggregation. The transcription rates of several previously unidentified genes either increased or decreased upon aggregation, while the transcription of other genes remained unchanged. The transcription rate of the N-CAM gene was 3.3-fold higher in dissociated than in aggregated CEB cells. This rate of transcription also increased when the brain tissue was dissociated into single cells and the increased rate was maintained by keeping the cells dissociated in the presence of Fab' fragments of antibodies to N-CAM. Decreased transcription rates of the N-CAM gene were also observed upon aggregation of P19 cells, a mouse embryonal carcinoma cell line. Primary chicken embryo liver cells, which aggregate primarily by calcium-dependent adhesion mechanisms, did not show changes in the N-CAM gene or in the other genes whose transcription rates changed in CEB cells and P19 cells. These observations suggest that the types of genes regulated by cell aggregation include those for CAMs themselves as well as for transcription factors that may control the expression of CAMs and other molecules significant for morphogenesis.