The single Dictyostelium chaperonin 60 gene, hspA, was cloned, sequenced and characterized. Sequence comparisons and a three-dimensional model for the structure of the encoded protein showed that it exhibits the conserved sequence and structural features expected for its role as the Dictyostelium mitochondrial chaperonin 60. Dictyostelium hspA contains two introns and, unusually for a member of this major heat shock gene family, is not stress-inducible in response to heat, cold or cadmium ions. Although transcription of hspA is down regulated during early Dictyostelium development in response to starvation, the levels of the chaperonin 60 protein remain constant throughout the life cycle. Consistent with the essential role of chaperonin 60 in mitochondrial biogenesis, we were unable to isolate mutants in which the hspA gene had been disrupted. However, transformants were isolated that exhibited differing levels of antisense inhibition of chaperonin 60 expression, depending upon the number of copies of the antisense-expressing plasmid in the genome. Orientation in phototaxis (and thermotaxis) was severely impaired in all antisense transformants, while growth and morphogenesis were markedly defective only in transformants with higher levels of antisense inhibition. This pattern of phenotypes is similar to that reported previously to result from targeted disruption of the mitochondrial large subunit rRNA gene in a subpopulation of mitochondria. This suggests that, regardless of the nature of the underlying genetic defect, mitochondrial deficiency impairs signal transduction more sensitively than other cellular activities.