We present with two members of a consanguineous Pakistani family with biochemical evidence of aminoacylase I deciency (ACY1D, MIM609924) yet disparate phenotypes. The proband (at 3 years) is profoundly hypotonic with severe developmental delay. Father, brother (8 years) and sister (9 years) are healthy with normal development. His mother has significant learning difficulties. The proband and his healthy brother excrete excess of N-acetylamino acids in the urine, characteristic of ACY1D. No excess of N-acetylamino acids was detected in plasma or CSF of the proband and ACY1 enzyme activity [EBV lymphoblasts (EBVL), fibrblasts, skeletal muscle, rectal muscle] gave in consistent results, reflecting low expression in these tissues. Western blotting revealed no detectable protein in samples from the two brothers and reduced protein in the sister, mother and father consistent with carrier status. Genomic sequencing of the ACY1 gene (all14 exons/exon-intron boundaries) failed to identify a mutation. ACY1 mRNA of normal length and amount was present in EBVL from all family members and in all tissues sampled from the proband. Sequencing of cDNA from a control and the proband showed 100% identity. To date a genetic cause of ACY1D has not been demonstrated in this family. Reports of ACY1 individuals existing as heterocomplex are lacking, although interaction with sphingosine kinase 1 has been described, raising the possibility of defective complex formation. This family adds to the speculation regarding the phenotype of ACY1D. more cases are required to ascertain whether this disorder has direct consequences, confers a risk of neurological sequelae or is simple a benign biochemical variant.