Brevetoxins resemble domoic acid in that these phycotoxins produce an autocrine excitotoxicity in rat cerebellar granule neurons. The resultant excessive activation of glutamate receptors produces a dysregulation of neuronal Ca²⁺ homeostasis that ultimately leads to cell death. In the present studies we have used FLIPR^TM to monitor real-time alterations in cytoplasmic Ca²⁺ levels in primary cultures of cerebellar granule neurons. Cerebellar granule neurons (CGNs) grown in 96-well plates were used for intracellular Ca²⁺ [Ca²⁺ ]_i measurements at day 10-13 in culture. Cells were loaded with the fluorescent Ca²⁺ indicator, Fluo-3 AM to monitor dynamic changes in [Ca²⁺ ]_i. Fluo-3AM is taken up by cells and entrapped intracellularly after hydrolysis to FLUO-3 by cytoplasmic esterases. FLIPR^TM possesses an automated 96- well pipettor which can be programmed to deliver fixed volumes of solutions simultaneously to all 96 culture wells from two separate 96-well source plates. Neurons were excited by the 488 nm line of the argon laser and Ca²⁺ -bound Fluo-3 emission in the 500-560 nm range was recorded with a CCD camera. Acute exposure to brevetoxins such as PbTx-1 produced a rapid and concentration dependent increase in intracellular [Ca²⁺ ]_i. MK-801 pretreatment produced a marked reduction in the integrated Fluo-3 fluorescence response to PbTx-1. Similarly, tetanus toxin reduced the integrated Fluo-3 fluorescence response to PbTx-1 as a consequence of its ability to inhibit glutamate release. Additional pharmacological analyses of the PbTx-1 response revealed that L-type Ca²⁺ channels and the reverse mode of operation of the Na⁺/ Ca²⁺ exchanger also contributed to the stimulated Ca²⁺ influx. These data document the utility of FLIPR^TM to delineate neuronal Ca²⁺ influx pathways associated with brevetoxin exposure.