Expression of the prespore-specific gene 3B in Dictyostelium discoideum Ax-2 cells is first detectable late in development with 3B mRNA levels peaking at 18 h (Corney et al., 1990). Sequence analysis of 3B cDNA and genomic clones revealed two exons, 319bp and 341bp long, separated by an 82bp intron, which encode a 219 residue protein with no significant similarity to any other reported gene product. Transcription starts at an A residue 45bp upstream from the translation initiation codon, preceded by a TATA-like sequence and an oligo-dT stretch. The 5' flanking sequence of the 3B gene is extremely A + T rich but contains five G/C rich stretches, each approximately 7bp long, which have strong sequence similarity to the G boxes found upstream of other developmentally regulated Dictyostelium genes. Analysis of both 3B promoter-CAT reporter gene and 3B promoter-lacZ reporter gene constructs showed that 908bp of 5' flanking sequence is sufficient to confer correct developmental and cell-type specific regulation. Sequential 5' deletion analysis revealed that positive elements lie upstream of position -304 and that negative element(s) lie between positions -264 and -241. Nevertheless, a 286bp promoter fragment containing only sequence located downstream of position -241 directed essentially correct reporter gene expression. Point mutation analysis identified cis-acting elements within this 'sufficient' promoter fragment which activate transcription (G box V and psp-AT type sequences). A short (56bp) region of the 3B promoter sequence containing both G box IV and the psp-AT type element binds two types of nuclear factor, one present in cells throughout development and a second that appears only in late development with a time course comparable to 3B gene induction.