Human calcitonin (CT) gene transcription is regulated by proximal 5' flanking sequences which mediate cAMP-induced expression, and by a distal basal enhancer region. Using transient expression of CT-CAT constructs, we showed that the basal enhancer is active in a CT-producing small cell lung cancer cell line (DMS53) and the thyroid C cell derived tumor line, TT, but is inactive in non-CT-producing cell lines. In deletional and direct mutational analyses of the distal enhancer region, disruption of two elements resembling recognition sequences for the helix-loop-helix (HLH) family of transcriptional regulatory proteins resulted in a significant loss of basal transcriptional enhancer action. These results suggest that HLH recognition motifs may mediate a significant portion of constitutive CT gene transcriptional activity in these cells. Nuclear protein extracts from DMS53 cells formed specific binding complexes with oligonucleotides containing two of these candidate enhancer sequences. However, proteins capable of binding to these CT gene HLH consensus recognition sites were not restricted to CT-producing cells. We conclude that members of the HLH protein family, some expressed ubiquitously and some expressed or activated in a tissue-restricted fashion, may combine to enhance CT gene transcription in tumor cells of neuroendocrine derivation.