The glial fibrillary acidic protein (GFAP) gene is alternatively spliced to give GFAP-α, the most abundant isoform, and seven other differentially expressed transcripts including GFAP-δ. GFAP-δ has an altered C-terminal domain that renders it incapable of self-assembly in vitro. When titrated with GFAP-α, assembly was restored providing GFAP-δ levels were kept low (∼10%). In a range of immortalized and transformed astrocyte derived cell lines and human spinal cord, we show that GFAP-δ is naturally part of the endogenous intermediate filaments, although levels were low (∼10%). This suggests that GFAP filaments can naturally accommodate a small proportion of assembly-compromised partners. Indeed, two other assembly-compromised GFAP constructs, namely enhanced green fluorescent protein (eGFP)-tagged GFAP and the Alexander disease–causing GFAP mutant, R416W GFAP both showed similar in vitro assembly characteristics to GFAP-δ and could also be incorporated into endogenous filament networks in transfected cells, providing expression levels were kept low. Another common feature was the increased association of αB-crystallin with the intermediate filament fraction of transfected cells. These studies suggest that the major physiological role of the assembly-compromised GFAP-δ splice variant is as a modulator of the GFAP filament surface, effecting changes in both protein– and filament–filament associations as well as Jnk phosphorylation.