Asexual development in Toxoplasma gondii is a vital aspect of the parasite's life cycle, allowing transmission and avoidance of the host immune response. Differentiation of rapidly dividing tachyzoites into slowly growing, encysted bradyzoites involves significant changes in both physiology and morphology. We generated microarrays of approximately 4,400 Toxoplasma cDNAs, representing a minimum of approximately 600 genes (based on partial sequencing), and used these microarrays to study changes in transcript levels during tachyzoite-to-bradyzoite differentiation. This approach has allowed us to (i) determine expression profiles of previously described developmentally regulated genes, (ii) identify novel developmentally regulated genes, and (iii) identify distinct classes of genes based on the timing and magnitude of changes in transcript levels. Whereas microarray analysis typically involves comparisons of mRNA levels at different time points, we have developed a method to measure relative transcript abundance between genes at a given time point. This method was used to determine transcript levels in parasites prior to differentiation and to further classify bradyzoite-induced genes, thus allowing a more comprehensive view of changes in gene expression than is provided by standard expression profiles. Newly identified developmentally regulated genes include putative surface proteins (a SAG1-related protein, SRS9, and a mucin-domain containing protein), regulatory and metabolic enzymes (methionine aminopeptidase, oligopeptidase, aminotransferase, and glucose-6-phosphate dehydrogenase homologues), and a subset of genes encoding secretory organelle proteins (MIC1, ROP1, ROP2, ROP4, GRA1, GRA5, and GRA8). This analysis permits the first in-depth look at changes in gene expression during development of this complex protozoan parasite.