Acute intermittent porphyria (AIP) results from a decreased activity of hepatic hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway. AIP is an autosomal dominant disorder with incomplete penetrance, characterized by acute neurovisceral attacks precipitated by several factors that induce the hepatic 5-aminolevulinic acid synthase, the first enzyme in the heme biosynthesis. Thus, a deficiency in HMBS activity results in an overproduction of porphyrin precursors and the clinical manifestation of the disease. Early diagnosis and counselling are essential to prevent attacks, and mutation analysis is the most accurate method to identify asymptomatic carriers in AIP families. In the present study, we have investigated the molecular defects in 55 unrelated Spanish patients with AIP, identifying 32 HMBS gene mutations, of which six were novel and ten were found in more than one patient. The novel mutations included a missense, an insertion, two deletions, and two splice site variants. Prokaryotic expression studies demonstrated the detrimental effect for the missense mutation, whereas reverse transcription-PCR and sequencing showed aberrant splicing caused by each splice site mutation. These results will allow for an accurate diagnosis of carriers of the disease in these families. Furthermore, they increase the knowledge about the molecular heterogeneity of AIP in Spain.