Amelogenin synthesis is initiated in a restricted time frame during odontogenesis. Polypeptides translated from several alternatively spliced isoforms of amelogenin mRNA have been identified in ameloblasts and odontoblasts. Recent studies suggest that the isoforms deleting exons 6a, 6b, and 6c produce polypeptides that might exert regulatory functions governing the late stages of ameloblast and odontoblast differentiation. Herein, the spatial and temporal expression of mouse amelogenin mRNA isoforms M194, M180, M73, and M59 have been determined around the perinatal development period using splice form-specific probes. Expression levels and distribution patterns varied with developmental stage and cell location. Amelogenin mRNA expression was most prominent within the enamel organ at boundaries between cell layers, beginning at the newborn stage (PN0.5). Odontoblasts supported the expression of M73 and M59 mRNA from developmental stages PN0.5 to PN1.5 (1 d of age). In contrast, ameloblasts expressed predominantly the M180 mRNA isoform with full exon 6 but devoid of exon 4. In the enamel organ, the stratum intermediun cells supported expression of the full-length isoform, M194, including the full exon 6 and exon 4 sequences, and strikingly, expression of M180 message was inhibited. In conclusion, ameloblasts, odontoblasts, and stratum intermedium cells demonstrate selective alternative splicing patterns of the amelogenin pre-mRNA transcript.