Antifreeze proteins (AFPs) are encoded by approximately 17 genes in the spruce budworm, Choristoneura fumiferana. Northern analysis using 6 different cDNA probes showed isoform-specific patterns that varied during development. Transcripts for the majority of isoforms were most abundant in the second instar overwintering stage, but some were also detected in first instar and even in egg stages. In situ hybridization using riboprobes corresponding to two 9 kDa protein isoforms showed differential AFP expression even in second instars; CfAFP10 RNA was detected in all tissues, but CfAFP337 RNA distribution was more limited. Two genomic regions encoding three AFP genes have been isolated. Presumptive regulatory regions conferred transcriptional activity when placed upstream of a luciferase reporter sequence and transfected into a C. fumiferana cell line. The CfAFP2.26 core promoter is an 87 bp sequence containing a TATA box, whereas the CfAFP2.7 core promoter is a 76 bp sequence with both a TATA box and CAAT box, which directed higher reporter activities when tested in vitro. Reporter activity was not enhanced with five different hormones, although lower activities were observed with all intron-containing constructs. AFP message half-life, as assessed using reporter assays, was not appreciably influenced by isoform-specific-3'UTRs. These studies successfully demonstrate the temporal and spatial diversity of AFP expression encoded by this small gene family, and underscore the complexity of their regulation.