Nacre or mother‐of‐pearl in the shell of Pinctada margaritifera is composed of 95–99% calcium carbonate and 1–5% organic matrix. In this study, we developed an original technique to characterize the genes differentially expressed in nacre‐forming cells (NFC) by combining suppression subtractive hybridization (SSH), to establish a cDNA subtractive library, with rapid amplification of cDNA ends (RACE)‐PCR. Seventy‐two specific cDNA sequences have been obtained so far. These include a protein containing two EF‐hand Ca2+‐binding domains which was completely sequenced after amplification by RACE‐PCR. Its specific expression as well as the specificity of the SSH method was confirmed by semi‐quantitative RT‐PCR on NFC and mantle cells.