Glycogen synthase kinase 3 α/β (GSK3α/β)were first identified over 25 years ago as as key enzymes in the regulation of carbohydrate metabolism. In their active form, these protein kinases catalyse inhibitory phosphorylations of glycogen synthase, thus cooperating in glycogen breakdown. However, subsequently, it has been recognised that they participate much more widely in the regulation of biological processes. GSK3α/β each contain a Tyr-residue [GSK3α(Tyr279)/GSK3β(Tyr216)], maturational intramolecular autophosphorylation of which facilitates and is necessary for activity. In terms of diurnal biological control, the most important regulatory event for GSK3 is thought to involve phosphorylation of GSK3α(Ser21)/GSK3β(Ser9) which inhibits their activity. However, there may also be additional, less well-defined sites of regulatory phosphorylations. Classically, phosphorylation of GSK3α(Ser21)/GSK3β(Ser9) is catalysed by protein kinase B/Akt (PKB/Akt), which is activated as a result of phosphatidylinositol 3-kinase (PI3K) signalling. Through this, insulin promotes glycogen synthesis by inhibiting GSK3, thus relieving the inhibition of glycogen synthase. In addition, activation of other protein kinases promotes phosphorylation of GSK3α(Ser21)/GSK3β(Ser9). Thus, activation of the ERK1/2 cascade leads to phosphorylation of GSK3α(Ser21)/GSK3β(Ser9), though this is probably mediated by ERK1/2-activated protein kinases rather than ERK1/2 themselves. Somewhat unexpectedly, in view of its role in stimulating glycogen breakdown, activation of protein kinase A may also promote phosphorylation of GSK3α(Ser21)/GSK3β(Ser9). A role for GSK3 (the emphasis was largely directed towards GSK3β) in the development of cardiac/cardiac myocyte hypertrophy was recently proposed by Morisco et al. (2000) and Haq et al. (2000). As reviewed by Shevstov et al. (2006), one current proposal is that that GSK3 in its active state mediates phosphorylation of β-catenin promoting its proteasomal degradation. When GSK3 is inhibited, β-catenin is stabilised and enters the nucleus where it derepresses the Groucho-mediated inhibition of Lef/TCF transcription factors and promotes transcription of the genes they regulate. Alternatively or additionally, GSK3-mediated phosphorylation of NFATs (specifically NFATc1) results in their retention in the cytoplasm (Beals et al. 1997). By either pathway, PI3K – PKB/Akt-mediated inhibition of GSK3 by powerful hypertrophic agonists such as endothelin-1 (ET-1) or phenylephrine could promote transcription of suitably-susceptible genes either by stabilising β-catenin or by promoting NFAT dephosphorylation by protein Ser/Thr phosphatases such as calcineurin. Thus GSK3 in its catalytically-active state [GSK3α(Ser21)/GSK3β(Ser9)dephosphorylated] is potentially antihypertrophic. We have recently re-examined aspects of this scheme in cardiac myocytes from neonatal rat hearts in primary culture. In outline, our conclusions are as follows. 1. Both GSK3 isoforms are expressed in cardiac myocytes, thus GSK3α should receive as much attention as GSK3β. 2. Insulin, which strongly promotes activation of PKB/Akt but which does not activate the ERK1/2 cascade in cardiac myocytes, causes an LY294002-sensitive phosphorylation of GSK3α(Ser21)/GSK3β(Ser9). This coincides with inhibition of GSK3 activity as assessed using an FPLC-based assay. However, we have never been able to detect any pro-hypertrophic effects (e.g increased cell area and myofibrillogenesis) of insulin. 3. ET-1 (which is at best a weak activator of PKB/Akt but is a strong activator of the ERK1/2 cascade) causes a U0126-sensitive phosphorylation of GSK3α(Ser21)/GSK3β(Ser9), implying involvement of the ERK1/2 cascade. 4. In contrast to the results with insulin and with the caveat of lack of specificity, the GSK3 inhibitor 1-azakenpaullone promotes an increase in myocyte area with an increase in the myocyte length/width ratio. Overall, we conclude that whilst there may be a role for GSK3 activity in the inhibition of the cardiac myocyte hypertrophic response (and a role for GSK3 inhibition in the development and continuation of the response), the situation is not as clearly defined as with other signalling pathways.