Homeostasis of the intracellular ionic concentration, in particular that of hydrogen ions, is pivotal to the maintenance of cell function and viability. Nonetheless, pH fluctuations in both the intracellular and the extracellular compartments can occurr during development, in physiological processes and in disease. The influence of pH variations on gene expression has been studied in different model systems, but only for a limited number of genes. We have performed a broad range analysis of the patterns of gene expression in normal human dermal fibroblasts at two different pH values (in the presence and in the absence of serum), with the aim of getting a deeper insight into the regulation of the transcriptional program as a response to a pH change. Using the Affymetrix gene chip system, we found that the expression of 2068 genes (out of 12 565) was modulated by more than two-fold at 24, 48 or 72 h after the shift of the culture medium pH to a more acidic value, stanniocalcin 1 being a remarkable example of a strongly up-regulated gene. Genes displaying a modulated pattern of expression included, among others, cell cycle regulators (consistent with the observation that acidic pH abolishes the growth of fibroblasts in culture) and relevant extracellular matrix (ECM) components. Extracellular matrix protein 2, a protein with a restricted pattern of expression in adult human tissues, was found to be remarkably overexpressed as a consequence of serum starvation. Since ECM components, whose expression is controlled by pH, have been used as targets for biomolecular intervention, we have complemented the Affymetrix analysis with a two-dimensional polyacrylamide gel electrophoresis analysis of proteins which are differentially secreted by fibroblasts at acidic or basic pH. Mass spectrometric analysis of more than 650 protein spots allowed the identification of 170 protein isoforms or fragments, belonging to 40 different proteins. Some proteins were only expressed at basic pH (including, for instance, tetranectin), while others (e.g., agrin) were only detectable at acidic pH. Some of the identified proteins may represent promising candidate targets for biomedical applications, e.g., for antibody-mediated vascular targeting strategies.