Studies were carried out to determine the possible mechanism(s) by which prolactin (PRL) stimulates alpha-lactalbumin and galactosyl transferase activity in cultured mouse mammary tissues. In earlier studies it was shown that the onset of the PRL stimulation of galactosyl transferase activity occurs between 4 and 8 hr after adding prolactin to cultured mouse mammary gland explants, and a maximum effect is evoked by 24 hr. In contrast, an effect of prolactin on alpha-lactalbumin activity occurred 24 hr after adding PRL to mammary gland cultures, but not at earlier times. In the present studies, it is shown that prolactin effects an increased tissue accumulation of mRNAs for both alpha-lactalbumin and galactosyl transferase after a 4 to 6 hr culture with prolactin. The lowest concentration of prolactin that stimulates the accumulation of both galactosyl transferase and alpha-lactalbumin mRNA is approximately 10 ng/ml, and a maximum response is achieved with 200 ng/ml. Thus, it is likely that the effect of PRL on the activity of galactosyl transferase is causally associated with the effect of prolactin on the accumulation of its mRNAs. However, the PRL stimulation of alpha-lactalbumin activity is not temporally associated with the tissue accumulation of the mRNA for alpha-lactalbumin.