CONTEXT: "Matricellular proteins" are a group of secreted, multifunctional extracellular matrix glycoproteins that includes thrombospondins (TSPs), tenascin-C (TNC), and SPARC (secreted protein acidic and rich in cysteine). They may be implicated in the dynamic developmental processes of the human fetal adrenal (HFA) where the outer, definitive zone (DZ) cells are postulated to proliferate, migrate centripetally, differentiate, and populate the inner, steroidogenic fetal zone (FZ). OBJECTIVE: Identify a matricellular molecule that likely plays a major role in HFA development. DESIGN: Studies involved RNA, cryosections, and cell cultures from 14-23 wk HFA's and human adult adrenal RNA. MAIN OUTCOME MEASURES: Transcripts encoding matricellular proteins, using real-time quantitative RT-PCR (qRT-PCR); SPARC localization, by immunostaining; ACTH regulation of SPARC expression and secretion, by qRT-PCR and Western blot. RESULTS: SPARC HFA mRNA was 100-fold, 700-fold, and 300-fold higher than TSP-1, TSP-2, and TNC mRNA, respectively. HFA SPARC mRNA was 3-fold higher than adult adrenals (P < 0.005), comparable to levels in adult brain (positive control), whereas mRNAs encoding TSP-1, TSP-2 were lower in fetal than adult adrenals. SPARC immunoreactivity was detected exclusively in the FZ, not DZ. ACTH, a key regulator of HFA growth and function, increased SPARC mRNA (by 1.7-fold at 1 nM, 48 h, P < 0.05) in isolated FZ cells, but not in DZ cells. ACTH up-regulation of SPARC protein was also detected in FZ cell lysates and culture medium. CONCLUSIONS: Results suggest a possible role for SPARC in development of functional and/or structural zonation of the HFA.