Rapidly labelled RNA containing poly(A) sequences has been isolated from polysomes of mouse parotid glands. Filtration on Millipore and binding to poly(u) immobilized on Sepharose-4B, together with significant RNase-resistance (11-16%) indicated that this polysomal fraction contains large tracts of polyadenylate sequences. A single injection of isoproterenol, a procedure that causes, after a lag period of 20 hrs, a marked stimulation of DNA synthesis and cell division in salivary glands of rodents, induces a biphasic increase in the incorporation of (3H)-adenosine into poly(A)-associated polysomal RNA. The incorporation of (3H)-adenosine is already increased within 1 hour after stimulation, decreases at 3 hrs and increases again at 8 hrs, parallelling previously reported changes on chromatin template activity of mouse parotids. However, the percent poly(A) content, and the size of the poly(A)-associated RNA remain constant. Actinomycin D given 30 min prior to isoproterenol, inhibits isoproterenol-Stimulated poly(A)-rich RNA synthesis. These results demonstrate that, as part of the early molecular events of its action, isoproterenol regulates transcription of adenylate-rich RNA in mouse parotid gland.