ICER corresponds to a group of alternatively spliced Inducible cAMP Early Repressors with high similarity, but multiple roles, including in circadian rhythm, and are involved in attenuation of cAMP-dependent gene expression. We present experimental and in silico data revealing biological differences between the isoforms with exon gamma (ICER) or without it (ICER gamma). Both isoforms are expressed in the liver and the adrenal glands and can derive from differential splicing. In adrenals the expression is circadian, with maximum at ZT12 and higher amplitude of ICER gamma. In the liver, the expression of ICER gamma is lower than Icer in the 24 h time frame. Icer mRNA has a delayed early response to forskolin. The longer ICER protein binds to three DNA grooves of the Per1 promoter, while ICER gamma only to two, as deduced by molecular modelling. This is in line with gel shift competition assays showing stronger binding of ICER to Per1 promotor. Only ICER gamma siRNA provoked an increase of Per1 expression. In conclusion, we show that ICER and ICER gamma have distinct biochemical properties in tissue expression, DNA binding, and response to forskolin. Data are in favour of ICER gamma as the physiologically important form in hepatic cells where weaker binding of repressor might be preferred in guiding the cAMP-dependent response. (C) 2020 The Authors. Published by Elsevier Inc.