Over the last decade, subtractive cloning approaches have been used extensively to isolate genes that are up- or down-regulated under various conditions. These techniques have provided the foundation for many subsequent studies concerning gene function and regulation and, as such, have been valuable tools for many biological fields. Over the past 10 years, we have used different subtractive cloning approaches to isolate genes in fish that are regulated in relation to hormonal stimulation or the stage of ovarian maturation. These include conventional cDNA subtraction followed by library screening, differential display PCR, suppression subtraction hybridization, and more recently, iterative PCR subtraction. We continue to use these techniques for the isolation of new genes involved in physiological processes in fish and bivalve molluscs. Examples that illustrate the use of these different subtractive cloning techniques are described, including where possible the advantages and disadvantages of each. In addition, the use of ancillary methods (e.g., “Reverse Northerns”) to facilitate the use of these subtractive approaches are discussed.