The main analytical use of Ca(2+)-regulated photoproteins from luminous coelenterates is for real-time non-invasive visualization of intracellular calcium concentration ([Ca(2+)]i) dynamics in cells and whole organisms. A limitation of this approach for in vivo deep tissue imaging is the fact that blue light emitted by the photoprotein is highly absorbed by tissue. Seven novel coelenterazine analogues were synthesized and their effects on the bioluminescent properties of recombinant obelin from Obelia longissima and aequorin from Aequorea victoria were evaluated. Only analogues having electron-donating groups (m-OCH3 and m-OH) on the C6 phenol moiety or an extended resonance system at the C8 position (1-naphthyl and α-styryl analogues) showed a significant red shift of light emission. Of these, only the α-styryl analogue displayed a sufficiently high light intensity to allow eventual tissue penetration. The possible suitability of this compound for in vivo assays was corroborated by studies with aequorin which allowed the monitoring of [Ca(2+)]i dynamics in cultured CHO cells and in hippocampal brain slices. Thus, the α-styryl coelenterazine analogue might be potentially useful for non-invasive, in vivo bioluminescence imaging in deep tissues of small animals.