This study examined the expression of metallothionein (MT) isoforms in the lungs of Lewis rats exposed to Cadmium (Cd) aerosols. With the use of isoform-specific oligonucleotide probes and Northern hybridization analysis, we demonstrated that a dramatic, rapid, and coordinate increase occurred in pulmonary MT-1 mRNA and MT-2 mRNA following Cd inhalation exposure. MT mRNAs levels reached a maximum at 2 h post-exposure and remained above control levels at 96 h after exposure. A considerable lag between the time of maximal elevations in MT mRNAs and MT protein accumulation was observed and suggested that regulatory mechanisms in addition to transcriptional control could be involved. MT expression (protein and mRNA) and Cd lung burden were directly related to aerosol Cd concentration. In situ hybridization and immunohistochemistry studies showed good correlation between the localization of MT protein and MT mRNAs. However, staining for MT protein and MT mRNA was not uniformly distributed in the lung. MT was particularly prominent within the alveolar compartment. Even within this area, however, heterogeneity of MT expression was evident. Experiments were subsequently conducted to determine whether prior exposure to Cd modulates the transcriptional activity of MT genes such that there is a greater elevation in gene expression upon reexposure to Cd. Surprisingly, animals pretreated with Cd exhibited a smaller incremental increase in MT mRNA levels in response to subsequent Cd exposure than controls with no prior treatment. Moreover, MT mRNA levels were elevated to a similar extent regardless of whether animals were exposed to Cd aerosols for 1, 2, or 3 weeks (3 h/day; 5 days/week). MT protein and lung Cd burden, on the other hand, exhibited an increasing linear trend as a function of exposure number. In summary, this study has demonstrated that: (1) the lung responds to Cd inhalation exposure by increasing MT mRNA and MT protein levels; (2) MT expression is prominent within alveolar cells but not all cells are MT positive; and (3) Cd-pretreatment does not increase the transcriptional potential of MT genes when the animal is subsequently reexposed to Cd.