We have assessed the relative contribution of Ca2+ entry and Ca2+ release from internal stores to the [Ca2+]i transients evoked by purinergic receptor activation in bovine adrenal chromaffin cells. The [Ca2+]i was recorded from single cells using ratiometric fura-2 microfluorometry. Two discrete groups of ATP-sensitive cells could be distinguished on the basis of their relative capacity to respond to ATP in the virtual absence of extracellular Ca2+. One group of cells (group I) failed to respond to ATP in the absence of Ca2+, was completely insensitive to UTP, and displayed suramin-blockable [Ca2+]i transients when challenged with ATP in the presence of external Ca2+. ATP activated a prominent and rapidly inactivating Mn2+ influx pathway in group I cells, as assessed by monitoring Mn2+ quenching of fura-2 fluorescence. In contrast, a second group of ATP-sensitive cells (group II) exhibited pronounced [Ca2+]i rises when challenged with ATP and UTP in the absence of Ca2+ and was completely insensitive to suramin. ATP and UTP activated a delayed and less prominent Mn2+ influx pathway in group II cells. Contrary to the nicotinic receptor agonist DMPP, which evoked a preferential release of epinephrine, ATP evoked a preferential release of norepinephrine, and UTP had no effect on secretion. Suramin nearly suppressed ATP-evoked norepinephrine release. We conclude that chromaffin cells contain two distinct and cell-specific purinoceptor subtypes. Although some cells express a P2U-type purinoceptor coupled to Ca2+ release from internal stores and to the associated slow Ca2+ refilling mechanism, other cells express a suramin-sensitive and UTP-insensitive purinoceptor exclusively coupled to Ca2+ influx, probably an ATP-gated channel. It is suggested that the ATP-gated channel is preferentially localized to norepinephrine-secreting chromaffin cells and supports specifically hormone output from these cells. Thus, the biochemical pathways involved in the exocytotic release of the two major stress-related hormones appear to be regulated by distinct signaling systems.