The effects of culture and membrane potential on Go alpha 39 expression were examined in neonatal rat cardiac myocytes. During six days of culture, the amount of Go alpha 39 in myocytes increased six-fold. The increase in Go alpha 39 appeared to be programmed, since Go alpha 39 of rat hearts also increased in vivo within three days after birth before declining by six days after birth. Furthermore, the age of the rat from which cardiac myocytes were isolated determined the amount of Go alpha 39 that accumulated in cultured cells with myocytes from two day-old rats producing more Go alpha 39 than myocytes from six day-old rats. In addition, agents which alter membrane potential (KCl and bupivacaine) inhibited the accumulation of Go alpha 39 in cultured myocytes. In an attempt to identify the signaling pathway in which cardiac Go alpha 39 is involved, muscarinic receptor-stimulated inositol phosphate production was examined, but was found to be comparable in myocytes that had six-fold differences in Go alpha 39 content. Thus Go alpha 39 does not appear to couple muscarinic receptors to phospholipase C in rat cardiac myocytes.