It is anticipated that γ-secretase inhibitors (γ-Sec-I) that modulate Notch processing will alter differentiation in tissues whose architecture is governed by Notch signaling. To explore this hypothesis, Han Wistar rats were dosed for up to 5 days with 10–100 μmol/kg b.i.d. γ-Sec-I from three chemical series that inhibit Notch processing in vitro at various potencies (Notch IC₅₀). These included an arylsulfonamide (AS) (142 nM), a dibenzazepine (DBZ) (1.7 nM), and a benzodiazepine (BZ) (2.2 nM). The DBZ and BZ caused dose-dependent intestinal goblet cell metaplasia. In contrast, the AS produced no detectable in vivo toxicity, despite higher exposure to free drug. In a time course using BZ, small intestinal crypt cell and large intestinal glandular cell epithelial apoptosis was observed on days 1–5, followed by goblet cell metaplasia on days 2–5 and crypt epithelial and glandular epithelial regenerative hyperplasia on days 4–5. Gene expression profiling of duodenal samples from BZ-dosed animals revealed significant time-dependent deregulation of mRNAs for various panendocrine, hormonal, and transcription factor genes. Somatostatin, secretin, mucin, CCK, and gastrin mRNAs were elevated twofold or more by day 2, and a number of candidate ‘early-predictive’ genes were altered on days 1–2, remaining changed for 4–5 days; these included Delta1, NeuroD, Hes1-regulated adipsin, and the Hes-regulated transcriptional activator of gut secretory lineage differentiation, the rat homolog of Drosophila atonal, Rath1. Western blotting of fecal protein from BZ-and DBZ-dosed animals exhibited increased levels of both anti-Rath1 reactive protein and anti-adipsin reactive proteins, confirming their potential value as noninvasive biomarkers of intestinal goblet metaplasia.