Extracellular matrix molecules—including chondroitin sulfate proteoglycans, hyaluronan, and tenascin‐R—are enriched in perineuronal nets (PNs) associated with subsets of neurons in the brain and spinal cord. In the present study, we show that similar cell type‐dependent extracellular matrix aggregates are formed in dissociated cell cultures prepared from early postnatal mouse hippocampus. Starting from the 5th day in culture, accumulations of lattice‐like extracellular structures labeled with Wisteria floribunda agglutinin were detected at the cell surface of parvalbumin‐expressing interneurons, which developed after 2–3 weeks into conspicuous PNs localized around synaptic contacts at somata and proximal dendrites, as well as around axon initial segments. Physiological recording and intracellular labeling of PN‐expressing neurons revealed that these are large fast‐spiking interneurons with morphological characteristics of basket cells. To study mechanisms of activity‐dependent formation of PNs, we performed pharmacological analysis and found that blockade of action potentials, transmitter release, Ca²⁺ permeable AMPA subtype of glutamate receptors or L‐type Ca²⁺ voltage‐gated channels strongly decreased the extracellular accumulation of PN components in cultured neurons. Thus, we suggest that Ca²⁺ influx via AMPA receptors and L‐type channels is necessary for activity‐dependent formation of PNs. To study functions of chondroitin sulfate‐rich PNs, we treated cultures with chondroitinase ABC that resulted in a prominent reduction of several major PN components. Removal of PNs did not affect the number and distribution of perisomatic GABAergic contacts but increased the excitability of interneurons in cultures, implicating the extracellular matrix of PNs in regulation of interneuronal activity. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007