Previous studies from this laboratory indicate that type-1 astroglia in primary culture are pharmacologically heterogeneous. Two competing hypotheses were proposed to explain the development of glial heterogeneity. First, that the heterogeneity may reflect stable subclasses of astroglia that express a set of receptor-signalling systems. Second, that astroglia can undergo qualitative changes in their expression of receptor-signalling systems with time in vitro. To distinguish between these two hypotheses, experiments were designed to examine neuroligand-evoked calcium responses within clones of type-1 astroglia. If stable and distinct subsets of astroglia were present, a clone derived from a single cell would exhibit uniform responses to a given set of neuroligands. Alternatively, if the pharmacological properties of astroglia underwent qualitative changes, astroglial clones should contain pharmacologically distinct cells. A video-based imaging system and the Ca2+ indicator dye Fura-2 were used to monitor receptor-mediated increases in Cai2+ upon receptor activation. Interestingly, only a fraction of the cells within a given clone responded to carbachol or histamine with an increase in Cai2+, whereas treatment with a P2Y purinergic receptor agonist generally increased Cai2+ in 100% of the cells within the clone. To examine the stability of the receptor signalling over time, individual astroglia within a number of clones were tested on different days for their ability to respond to neuroligands. The results of these experiments indicated that individual astroglial cells tended to lose their responsiveness to certain ligands such as carbachol and histamine as they developed responsiveness to others such as norepinephrine. Our data indicate that during development neurotransmitter receptors on astroglial cells are regulated by both internal and external mechanisms. Glial proliferation produces a variety of pharmacologically distinct astroglial cells. Exposure to neurotransmitters can qualitatively turn off some, but not all, astroglial receptor systems.