The a2 splice variant of the TRPM3 gene encodes a Ca²⁺ permeable channel. This allows studying the activation and inhibition of this channel with fluorescent Ca²⁺-indicators. TRPM3a2 channels display the unusal property of becoming highly Ca²⁺ permeable in the absence of extracellular Na⁺ even without agonist stimulation. We exploited this property and devised a protocol that allowed testing a given substance for agonistic as well as antagonistic properties in the same experiment. We validated this approach with 1 mM Gd³⁺, a known blocker of TRPM3 channels, and found Ca²⁺ influx through TRPM3a2 channels to be suppressed completely under our conditions. However, blockers of voltage dependent Ca²⁺-channels (verapamil and nimodipine) were without effect, even at high concentrations. Since TRPM3 is expressed in human and rodent brain, we tested catecholamine and monoamine neurotransmitters, but found neither agonistic nor antagonistic effects. Equally, L-amino acids (including the neurotransmitters) had no effects on TRPM3a2 mediated Ca²⁺ fluxes. However, steroidal compounds strongly activate TRPM3a2 channels. We confirmed the agonistic effects of these substances and their specificity electrophysiologically.