Abstract Cost and availability have often dictated the use of heterologous/alien prolactins in experiments, particularly in vivo. The assumption has been that what is initiated in the target cell is representative of the homologous hormone since many heterologous mammalian prolactins bind to and activate rodent receptors. Here, we examined gene expression in mouse liver in response to a 7‐day treatment with recombinant mouse prolactin (mRecPRL), recombinant ovine prolactin (oRecPRL) and pituitary extract ovine prolactin (oPitPRL). Having established mouse ribosomal protein S9 as the most stable reference gene in the liver in the absence and presence of prolactin treatment, we examined expression of the two most highly expressed prolactin receptors (PRLRs) and three members of the Cyp3a group of cytochrome P450 isoenzymes by qRTPCR. For short form (SF) 3 PRLR, mRecPRL doubled expression while for oRecPRL and oPitPRL expression was only 1.3‐fold control. For the long form (LF) PRLR, changes were similar to those seen for SF 3 PRLR, such that the SF3:LF PRLR ratio remained the same. Expression of the Cyp3as was also dependent on the prolactin origin and, although mRecPRL always stimulated, the other PRLs caused varying results. Compared to control, Cyp3a16 was stimulated 12‐fold by mRecPRL, 3‐fold by oRecPRL, and 6‐fold by oPitPRL. For Cyp3a41, mRecPRL was 3.7‐fold control, oRecPRL was without effect, and oPitPRL was 2‐fold control. Importantly, for Cyp3a44, mRecPRL stimulated 2‐fold, whereas both oRecPRL and oPitPRL had an opposite, inhibitory effect, with expression at 0.5‐fold control. We conclude that homologous hormone had the largest stimulatory effect on expression of all measured genes and that by contrast heterologous hormone showed reduced activity, no activity, or opposite activity, depending on the gene being analyzed. Thus, experimentation using alien heterologous PRL may lead to inaccurate conclusions.