Growing evidence indicates that alternative or aberrant pre-mRNA splicing takes place during the development, progression, and metastasis of breast cancer. However, which splicing changes that might contribute directly to tumorigenesis or cancer progression remain to be elucidated. We used splicing-sensitive microarrays to detect differences in alternative splicing between two breast cancer cell lines, MCF7 (estrogen receptor positive) and MDA-MB-231 (estrogen receptor negative), as well as cultured human mammary epithelial cells. Several splicing alterations in genes, including CD44, FAS, RBM9, hnRNPA/B, APLP2, and MYL6, were detected by the microarray and verified by reverse transcription-PCR. We also compared splicing in these breast cancer cells cultured in either two-dimensional flat dishes or in three-dimensional Matrigel conditions. Only a subset of the splicing differences that distinguish MCF7 cells from MDA-MB-231 cells under two-dimensional culture condition is retained under three-dimensional conditions, suggesting that alternative splicing events are influenced by the geometry of the culture conditions of these cells. Further characterization of splicing patterns of several genes in MCF7 cells grown in Matrigel and in xenograft in nude mice shows that splicing is similar under both conditions. Thus, our oligonucleotide microarray can effectively detect changes in alternative splicing in different cells or in the same cells grown in different environments. Our findings also illustrate the potential for understanding gene expression with resolution of alternative splicing in the study of breast cancer.