A subset of breast cancer patients (14% of our data set) neoadjuvantly treated with epirubicin-based chemotherapy experience complete remission associated with prolonged survival. Based on massively parallel transcriptome analysis, we describe for the first time early in situ effects of epirubicin-based chemotherapy in a neoadjuvant setting. We took jet-needle biopsies from 22 individual tumors immediately before and 24 h after the first cycle of chemotherapy. Clontech's 1.2 human cancer complementary DNA arrays were used. Jet-needle biopsies (30 mm3) yielded sufficient material (20–60 μg total RNA) for gene expression profiling. Of 1,185 genes selected for the array 30–50% were expressed. The most prominent early effect of chemotherapy was an upregulation of the cyclin kinase inhibitor WAF1, an important regulator of the G1–S transition in 82% of the tumors (average induction 2.8-fold; range 1.5- to 7-fold). Of the other cyclin kinase inhibitors, only p16-INK4A and p57-KIP2 were upregulated in a small subset of tumors. We observed a moderate chemotherapy-induced upregulation of various DNA repair enzymes. This group included XPC, an initiator of global genome nucleotide excision repair; endonuclease III homologue 1 (HNTH1) and DNA-damage-inducible transcript 3 (DDIT3, GADD153). Preliminary supervised cluster analysis seems to be encouraging for response prediction. Jet-needle technology is appropriate for in situ response monitoring in neoadjuvantly treated breast cancer patients. Chemotherapy-induced effects on cell-cycle control and DNA repair could be demonstrated as early as 24 hrs after the onset of therapy.